Novel polypeptide and its DNA

ABSTRACT

It is intended to construct a screening method, etc. for searching for compounds which are capable of binding to apolipoprotein A-I and thus promote or inhibit the binding of a cubulin fragment to apolipoprotein A-I, and provide preventives or remedies containing compounds obtained by the above screening method, etc. These compounds, etc. are usable in preventives and remedies for various diseases in which cubulin participates and the like.

TECHNICAL FIELD

The present invention relates to partial peptides of cubilin and theirfunctions.

BACKGROUND ART

The level of HDL-cholesterol [cholesterol contained in HDL (high densitylipoprotein); hereinafter, sometimes abbreviated to “HDL-C”] is known asa negative risk factor in coronary artery diseases. Various drugs hasbeen developed for the purpose of raising that level, but thus developeddrugs, such as fibrate type drugs, are not sufficient though they raiseHDL-C levels significantly. Besides, such rise in HDL-C levelsrepresents a consequential factor. Recent studies using transgenic mice,etc. have revealed that, as long as the true purpose is to extractcholesterol from foci of arteriosclerosis, what is needed is technologyto raise the level of apolipoprotein A-I (hereinafter, sometimesabbreviated to “apo A-I”) that plays a major role in the cholesterolextraction.

Apo A-I in plasma exists for the most part as HDL in the form ofcomplexes with phospholipid, cholesterol, cholesterol ester and so on.These complexes are catabolized mainly in the liver. However, it isknown that in some diseases such as hypertriglyceridemia, HDL particleswith increased triglyceride (hereinafter, sometimes abbreviated to “TG”)contents are converted into smaller apo A-I particles by various lipasesand then catabolized mainly in the kidney. The catabolism in the kidneyis achieved as follows: small particles of apo A-I that have beenfiltered through the uriniferous tubule are re-absorbed in the proximaltubule and then degraded. Kozyraki et al. have been reported thatcubilin (GenBank Accession No. AF034611) molecules expressed on theuriniferous tubule epithelium and known to bind to vitamin B₁₂-intrinsicfactor complex and LDL receptor-related protein (LRP)-associated protein(hereinafter, sometimes abbreviated to “RAP”) also bind to apo A-I (see,for example, Nature Medicine Vol. 5, No. 6, 656-661 (1999); Proc. Natl.Acad. Sci. USA, Vol. 96, 10158-10163 (1999)). However, relations betweenthe binding of cubilin to apo A-I and plasma apo A-I or HDLconcentration, or relations between this binding and arteriosclerosishave not yet been elucidated.

The binding of cubilin to apo A-I may be an important finding for thedevelopment of therapeutics for hypo-high density lipoproteinemia, whichis a problem in diseases such as high hypertriglyceridemia, and for thedevelopment of drugs for treating arteriosclerosis at the root. Tospecify the apo A-I-binding domain in cubilin molecules is extremelyuseful, for example, for developing anti-cubilin-apo A-I-binding domainantibody and screening for certain low molecular weight compounds.

Thus, the finding that the apo A-I-binding site is located in a specificregion in cubilin molecules is extremely important in view of clinicalapplication, and the identification of this binding site has beeneagerly awaited. Furthermore, the raising of serum apo A-I concentrationand HDL concentration by administering a substance that inhibits thebinding of apo A-I to cubilin partial fragments, and therapeutics forarteriosclerosis comprising such a substance have been eagerly awaitedin order to treat hypertriglyceridemia, hypercholesterolemia andarteriosclerotic diseases.

DISCLOSURE OF THE INVENTION

The present invention provides the identification of the apo A-I-bindingsite on cubulin; the construction of a screening method for a compoundthat inhibits the binding of a cubilin fragment comprising theidentified binding site to apo A-I; and a medicine comprising a compoundobtained by the constructed screening method.

To specify the apo A-I-binding site in cubilin molecules leads toclinical application of apo A-I-binding fragments of cubilin per se,antibodies thereto, and low molecular weight compounds that inhibit thebinding of cubilin figments to apo A-I.

As a result of intensive and extensive researches, the present inventorshave found apo A-I-binding activity in the CUB7-CUB14 fragment peptide(Blood 91, 3593-3600 (1998)) and the CUB9-CUB14 fragment peptide ofcubilin. Then, the inventors have developed an experimental system formeasuring apo A-I-binding using these fragments. Based on thesefindings, the inventors have made further investigations. Thus, thepresent invention has been achieved.

The present invention relates to:

-   -   (1) A polypeptide having the ability to bind to apolipoprotein        A-I which is a partial fragment of cubilin, an amide or ester of        said polypeptide, or a salt of said polypeptide,    -   (2) The polypeptide, amide or ester thereof, or salt thereof        of (1) above, wherein the polypeptide is characterized by having        an amino acid sequence identical or substantially identical with        the amino acid sequence as shown in SEQ ID NO: 10,    -   (3) The polypeptide, amide or ester thereof, or salt thereof        of (1) above, wherein the polypeptide has the amino acid        sequence as shown in SEQ ID NO: 10,    -   (4) The polypeptide, amide or ester thereof, or salt thereof        of (1) above, wherein the polypeptide is characterized by having        an amino acid sequence identical or substantially identical with        the amino acid sequence as shown in SEQ ID NO: 19,    -   (5) The polypeptide, amide or ester thereof, or salt thereof        of (1) above, wherein the polypeptide has the amino acid        sequence as shown in SEQ ID NO: 19,    -   (6) A DNA comprising a DNA encoding the polypeptide of (1)        above,    -   (7) The DNA of (6) above, wherein the DNA comprises the base        sequence as shown in SEQ ID NO: 9,    -   (8) The DNA of (6) above, wherein the DNA comprises the base        sequence as shown in SEQ ID NO: 22,    -   (9) A recombinant vector comprising the DNA of (6) above,    -   (10) A transformant transformed with the recombinant vector        of (9) above,    -   (11) A method of producing the polypeptide, amide or ester        thereof, or salt thereof of (1) above, which is characterized by        culturing the transformant of (10) above and allowing the        transformant to produce the polypeptide,    -   (12) An antibody to the polypeptide, amide or ester thereof, or        salt thereof of (1) above,    -   (13) A method of screening for compounds or salts thereof that        promote or inhibit the activity of the polypeptide or salt        thereof of (1) above, wherein the method is characterized by        using the polypeptide, amide or ester thereof, or salt thereof        of (1) above,    -   (14) A screening kit for compounds or salts thereof that promote        or inhibit the activity of the polypeptide, amide or ester        thereof, or salt thereof of (1) above, wherein the kit comprises        the polypeptide or salt thereof of (1) above,    -   (15) A compound or salt thereof that promotes or inhibits the        activity of the polypeptide, amide or ester thereof, or salt        thereof of (1) above, wherein said compound or salt thereof is        obtainable by using the screening method of (13) above or the        screening kit of (14) above,    -   (16) A medicine comprising a compound or salt thereof that        promotes or inhibits the activity of the polypeptide, amide or        ester thereof, or salt thereof of (1) above, wherein said        compound or salt thereof is obtainable by using the screening        method of (13) above or the screening kit of (14) above,    -   (17) A prophylactic and/or therapeutic agent for renal        disorders, nephritis, nephropathy, proteinuria, nervous        disorders or vitamin B₁₂ deficiency, comprising a compound or        salt thereof that promotes or inhibits the activity of the        polypeptide, amide or ester thereof, or salt thereof of (1)        above, wherein said compound or salt thereof is obtainable by        using the screening method of (13) above or the screening kit        of (14) above,    -   (18) A prophylactic and/or therapeutic agent for hyperlipemia,        hypertriglyceridemia, hypo-high density lipoproteinemia,        hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes,        obesity, arteriosclerosis, myocardial infarction or angina,        comprising a compound or salt thereof that promotes or inhibits        the activity of the polypeptide, amide or ester thereof, or salt        thereof of (1) above, wherein said compound or salt thereof is        obtainable by using the screening method of (13) above or the        screening kit of (14) above,    -   (19) A medicine comprising the polypeptide, amide or ester        thereof, or salt thereof of (1) above,    -   (20) A prophylactic and/or therapeutic agent for diabetes,        obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia,        hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or        nervous disorders, comprising the polypeptide, amide or ester        thereof, or salt thereof of (1) above,    -   (21) A diagnostic agent that is characterized by the use of the        DNA of (6) above,    -   (22) A diagnostic agent comprising the antibody of (12) above,    -   (23) A method for preventing and/or treating renal disorders,        nephritis, nephropathy, proteinuria, nervous disorders or        vitamin B₁₂ deficiency in a mammal, comprising a compound or        salt thereof that promotes or inhibits the activity of the        polypeptide, amide or ester thereof, or salt thereof of (1)        above, wherein said compound or salt thereof is obtainable by        using the screening method of (13) above or the screening kit        of (14) abvove,    -   (24) A method for preventing and/or treating hyperlipemia,        hypertriglyceridemia, hypo-high density lipoproteinemia,        hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes,        obesity, arteriosclerosis, myocardial infarction or angina,        characterized by administering to a mammal an effective amount        of a compound or salt thereof that promotes or inhibits the        activity of the polypeptide, amide or ester thereof, or salt        thereof of (1) above, wherein said compound or salt thereof is        obtainable by using the screening method of (13) above or the        screening kit of (14) above,    -   (25) Use of a compound or salt thereof that promotes or inhibits        the activity of the polypeptide, amide or ester thereof, or salt        thereof of (1) above, for manufacturing a prophylactic and/or        therapeutic agent for hyperlipemia, hypertriglyceridemia,        hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I,        after meal hyperlipemia, diabetes, obesity, arteriosclerosis,        myocardial infarction or angina, wherein said compound or salt        thereof is obtainable by using the screening method of (13)        above or the screening kit of (14) above,    -   (26) A method for preventing and/or treating diabetes, obesity,        arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high        density lipoproteinemia, hypoapolipoproteinemia A-I or nervous        disorders, characterized by administering to a mammal an        effective amount of the polypeptide, amide or ester thereof, or        salt thereof of (1) above, and    -   (27) Use of the polypeptide, amide or ester thereof, or salt        thereof of (1) above for manufacturing a prophylactic and/or        therapeutic agent for diabetes, obesity, arteriosclerosis,        hyperlipemia, hypertriglyceridemia, hypo-high density        lipoproteinemia, hypoapolipoproteinemia A-I or nervous        disorders.

Further, the present invention provides:

-   -   (28) The polypeptide, amide or ester thereof, or salt thereof        of (2) or (4) above, wherein the amino acid sequence        substantially identical with the amino acid sequence as shown in        SEQ ID NO: 10 or SEQ ID NO: 19 is an amino acid sequence having        about 50% or more (preferably about 60% or more, more preferably        about 70% or more, still more preferably about 80% or more,        especially preferably about 90% or more, and most preferably        about 95% or more) homology to the amino acid sequence as shown        in SEQ ID NO: 10 or SEQ ID NO: 19, and    -   (29) The polypeptide, amide or ester thereof, or salt thereof        of (2) or (4) above, wherein the amino acid sequence        substantially identical with the amino acid sequence as shown in        SEQ ID NO: 10 or SEQ ID NO: 19 is (i) an amino acid sequence        wherein one or more amino acids (preferably about 1-30 amino        acids) are deleted from the amino acid sequence as shown in SEQ        ID NO: 10 or SEQ ID NO: 19; (ii) an amino acid sequence wherein        one or more amino acids (preferably about 1-30 amino acids) are        added to the amino acid sequence as shown in SEQ ID NO: 10 or        SEQ ID NO: 19; (iii) an amino acid sequence wherein one or more        amino acids (preferably about 1-30 amino acids) amino acids are        substituted with other amino acids in the amino acid sequence as        shown in SEQ ID NO: 10 or SEQ ID NO: 19; or (iv) an amino acid        sequence which is a combination of these sequences.

Further, the DNA of the invention and the polypeptide, amide or esterthereof, or salt thereof of the invention may be utilized in basicresearches such as molecular markers, tissue markers, chromosomemapping, identification of genetic diseases or designing of primers,probes, and so on.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of a human cubilin mature protein(to be continued to FIG. 2).

FIG. 2 shows the amino acid sequence of a human cubilin mature protein(continued from FIG. 1).

FIG. 3 shows the results of Western blotting carried out in Example 2.In this Figure, lane C shows pSecTag2-transfected COS7 cells; lanes Iand II show COS7 cells transfected with expression vectors comprisingpSecTag2 plasmids into which cDNAs encoding human cubilin partialfragments I and II are incorporated, respectively; lane III shows theculture supernatant from pTB2116-transfected COS cells; and lanes IV andV show COS7 cells transfected with expression vectors comprisingpSecTag2 plasmids into which cDNAs encoding human cubilin partialfragments IV and V are incorporated, respectively. “Intracellular”denotes PVDF membranes blotted from those gels on which disruptedmaterials from individual cells were electrophoresed, and “CultureSupernatant” denotes PVDF membranes blotted from those gels on whichculture supernatants from individual cells were electrophoresed.

FIG. 4 shows the results of Western blotting carried out in Example 3.In this Figure, lanes I to V show culture supernatants containingpartial fragments I to V, respectively, and lane M shows molecularmarkers. “Elution” denotes PVDF membranes blotted from those gels onwhich EDTA-selectively eluted fractions were electrophoresed, and “Pass”denotes PVDF membranes blotted from those gels on which non-bindingfractions were electrophoresed.

FIG. 5 shows the results of Western blotting carried out in Examples 4and 5.

The utmost left lane in each blotting shows the migration of molecularmarkers.

Mock, Cub7-12, Cub9-14, Cub7-14 and Cub13-20 represent vectorstransfected into COS7 cells. Mock means a vector alone. Cub7-12 means avector expressing a cDNA encoding the CUB7-CUB12 fragment peptide, andCub9-14 means a vector expressing a cDNA encoding the CUB9-CUB14fragment peptide. Cub7-14 corresponds to the fragment III described inExample 3, and Cub13-20 corresponds to the fragment IV described inExample 3.

In this Figure, “Sample” denotes sample solutions applied to humanapolipoprotein A-I-binding resin; “Column” denotes fractions passedthrough columns; “Wash” denotes fractions eluted at the time of washing;and “Elution” denotes fractions eluted by EDTA. In the “Pass” panel, thebands appearing at approx. 70 kDa are considered to be non-specificrecognition bands attributable to high concentration albumin.

FIG. 6 shows the results of Western blotting (A) and CBB staining (B)carried out in Example 6. The numbers of individual lanes correspond tothe Nos. of fractions eluted with 10 mM-200 mM imidazole gradient inExample 6.

FIG. 7 shows the biotin-added human apolipoprotein A-I-binding curveobtained in Example 7. In this Figure, “FIU” denotes valuesquantitatively determined with a fluorescence measuring apparatus.

BEST MODE FOR CARRYING OUT THE INVENTION

The polypeptide of the invention that is a partial fragment of cubilinhaving the ability to bind to apolipoprotein A-I (hereinafter, sometimessimply referred to as the “polypeptide of the invention”) may be apolypeptide derived from cells of human or other warm-blooded animals(e.g. guinea pig, rat, mouse, chicken, rabbit, pig, sheep, bovine, ormonkey), such as hepatocyte, splenocyte, nerve cell, glia cell,pancreatic β cell, bone marrow cell, mesangial cell, Langerhans' cell,epidermic cell, epithelial cell, endothelial cell, fibroblast,fibrocyte, myocyte, fat cell, immune cell (e.g. macrophage, T cell, Bcell, natural killer cell, mast cell, neutrophil, basophil, eosinophil,monocyte), megakaryocyte, synovial cell, cartilage cell, bone cell,osteoblast, osteoclast, mammary gland cell or interstitial cell; orprecursor cells, stem cells or cancer cells of these cells; or may be apolypeptide derived from any of the tissues where such cells arepresent, such as brain, any region thereof (e.g. olfactory bulb,amygdaloid nucleus, basal ganglia, hippocampus, thalamus, hypothalamus,cerebral cortex, medulla, or cerebellum), spinal cord, pituitary,stomach, pancreas, kidney, liver, gonad, thyroid, gall-bladder, bonemarrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (e.g.large intestine and small intestine), blood vessel, heart, thymus,spleen, salivary gland, peripheral blood, prostate, testis, ovary,placenta, uterus, bone, cartilage, joint, skeletal muscle, etc.Alternatively, the polypeptide of the invention may be a recombinantpolypeptide or a synthetic polypeptide.

When the polypeptide of the invention has a signal peptide, thepolypeptide may be efficiently secreted out of cells.

The partial fragment of cubilin having the ability to bind toapolipoprotein A-I may be any partial peptide as long as it is a partialpeptide of cubilin and has the ability to bind to apolipoprotein A-I.Preferably, the partial peptide is a polypeptide having an amino acidsequence identical or substantially identical with the amino acidsequence as shown in SEQ ID NO: 10 or a polypeptide having an amino acidsequence identical or substantially identical with the amino acidsequence as shown in SEQ ID NO: 19.

As an amino acid sequence substantially identical with the amino acidsequence as shown in SEQ ID NO: 10, an amino acid sequence having about50% or more, preferably about 60% or more, more preferably about 70% ormore, still more preferably about 80% or more, especially preferablyabout 90% or more, and most preferably about 95% or more homology to theamino acid sequence as shown in SEQ ID NO: 10 may be given.

As an amino acid sequence substantially identical with the amino acidsequence as shown in SEQ ID NO: 19, an amino acid sequence having about50% or more, preferably about 60% or more, more preferably about 70% ormore, still more preferably about 80% or more, especially preferablyabout 90% or more, and most preferably about 95% or more homology to theamino acid sequence as shown in SEQ ID NO: 19 may be given.

As a polypeptide having an amino acid sequence substantially identicalwith the amino acid sequence as shown in SEQ ID NO: 10, such apolypeptide is preferable that has the above-described amino acidsequence substantially identical with the amino acid sequence as shownin SEQ ID NO: 10 and yet has a substantially identical nature with anature of a polypeptide having the amino acid sequence as shown in SEQID NO: 10.

As a polypeptide having an amino acid sequence substantially identicalwith the amino acid sequence as shown in SEQ ID NO: 19, such apolypeptide is preferable that has the above-described amino acidsequence substantially identical with the amino acid sequence as shownin SEQ ID NO: 19 and yet has a substantially identical nature with anature of a polypeptide having the amino acid sequence as shown in SEQID NO: 19.

As a substantially identical nature, a nature to bind to apolipoproteinA-I may be given, for example. The term “substantially identical” meansthat such a nature is qualitatively identical. Therefore, it ispreferable that a nature such as apolipoprotein A-I-binding ability isidentical (e.g. about 0.1-to about 100-fold, preferably about 0.5-toabout 10-fold, more preferably about 0.5- to about 2-fold), butquantitative factors such as the extent of that nature, the molecularweight of the polypeptide, etc. may be different.

More specifically, examples of the polypeptide comprising an amino acidsequence substantially identical with the amino acid sequence as shownin SEQ ID NO: 10 or SEQ ID NO: 19 include polypeptides (the so-calledmuteins) comprising (i) an amino acid sequence wherein one or more aminoacids (preferably about 1-30, more preferably about 1-10, still morepreferably several (1-5) amino acids) are deleted from the amino acidsequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (ii) an amino acidsequence wherein one or more amino acids (preferably about 1-30, morepreferably about 1-10, still more preferably several (1-5) amino acids)are added to the amino acid sequence as shown in SEQ ID NO: 10 or SEQ IDNO: 19; (iii) an amino acid sequence wherein one or more amino acids(preferably about 1-30, more preferably about 1-10, still morepreferably several (1-5) amino acids) are inserted into the amino acidsequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; (iv) an amino acidsequence wherein one or more amino acids (preferably about 1-30, morepreferably about 1-10, still more preferably several (1-5) amino acids)amino acids are substituted with other amino acids in the amino acidsequence as shown in SEQ ID NO: 10 or SEQ ID NO: 19; or (v) an aminoacid sequence which is a combination of these sequences.

When the amino acid sequence has insertion(s), deletion(s) orsubstitution(s) as described above, the positions of such insertion(s),deletion(s) or substitution(s) are not particularly limited. However,such positions are preferably those which are not essential for the apoA-I-binding in the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:19.

Throughout this specification, the left end of each polypeptide is itsN-terminus, and the right end thereof is its C-terminus according to theconvention in peptide description. The C-terminus of the polypeptide ofthe invention (such as a polypeptide comprising the amino acid sequenceas shown in SEQ ID NO: 10) is usually a carboxyl group (—COOH) or acarboxylate (—COO), but it may be an amide (—CONH₂) or an ester (—COOR).

Examples of R of the above ester group include C₁₋₆ alkyl groups (e.g.methyl, ethyl, n-propyl, isopropyl or n-butyl), C₃₋₈ cycloalkyl groups(e.g. cyclopentyl or cyclohexyl), C₆₋₁₂ aryl groups (e.g. phenyl orα-naphthyl), C₇₋₁₄ aralkyl groups such as phenyl-C₁₋₂ alkyl groups (e.g.benzyl or phenethyl) and α-naphthyl-C₁₋₂ alkyl groups (e.g.α-naphthylmethyl). In addition, the ester group also includespivaloyloxymethyl esters that are universally used as oral esters.

When the polypeptide of the invention has a carboxyl group (orcarboxylate) at any position other than its C-terminus, such apolypeptide that the carboxyl group may be amidated or esterified isalso included in the polypeptide of the invention. The ester in thiscase may be, for example, any of the esters mentioned above for theC-terminal ester.

Furthermore, the polypeptide of the present invention includes thosepolypeptides in which the N-terminal amino acid residue (e.g. Met) isprotected by a protective group (e.g. C₁₋₆ acyl group such as C₁₋₆alkanoyl group (e.g. formyl group or acetyl group)); those polypeptidesin which the N-terminal Glu generated through in vivo cleavage ispyroglutaminated; those polypeptides in which a substituent on a sidechain of an amino acid (e.g. OH, SH, amino group, imidazole group,indole group, or guannidino group) is protected by an appropriateprotective group (e.g. C₁₋₆ acyl group such as C₁₋₆ alkanoyl group (e.g.formyl group or acetyl group)); and conjugated polypeptides such as theso-called glycopolypeptides to which sugar chains are linked.

As the salt of the polypeptide of the invention, salts formed withphysiologically acceptable acids (e.g. organic or inorganic acids) orbases (e.g. alkali metals) are used. Especially preferable arephysiologically acceptable acid addition salts. Examples of such saltsinclude salts formed with inorganic acids (e.g. hydrochloric acid,phosphoric acid, hydrobromic acid or sulfuric acid) and salts formedwith organic acids (e.g. acetic acid, formic acid, propionic acid,fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid,malic acid, oxalic acid, benzoic acid, methanesulfonic acid orbenzenesulfonic acid).

The polypeptide or salt thereof of the present invention can be producedfrom the afore-mentioned cells or tissues of human or other warm-bloodedanimals by publicly known purification methods for polypeptides(proteins). Alternatively, the polypeptide of the invention can beproduced by culturing a transformant described later comprising a DNAencoding the polypeptide. It can also be produced in accordance with theprocedures for peptide synthesis which are described later.

When the polypeptide of the invention is produced from tissues or cellsof human or other warm-blooded animals, the relevant tissue or cell ishomogenized and then the polypeptide of the present invention isextracted with acids, etc. The polypeptide can be purified and isolatedfrom the resultant extract by a combination of chromatography, such asreversed phase chromatography, ion exchange chromatography and so on.

For the synthesis of the polypeptide of the invention or salt or amidethereof, any of the commercially available resins for polypeptidesynthesis may be used. Examples of such resins include chloromethylresin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin,4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAMresin, 4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamideresin, 4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin, and4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin. Using such aresin, amino acids appropriately protected at their α-amino groups andside chain functional groups are condensed on the resin according to theamino acid sequence of the polypeptide of interest by publicly knowncondensation methods. At the final stage of the reaction, all protectivegroups are removed simultaneously with the cleavage of the polypeptidefrom the resin. Then, in a highly diluted solution, intramoleculardisulfide bond formation reaction is carried out to obtain thepolypeptide of interest or amide thereof.

With respect to the above-described condensation of protected aminoacids, various activators useful for polypeptide synthesis may beutilized. Among all, carbodiimide reagents are especially preferred.Examples of carbodiimide reagents include DCC,N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide. For activation by thesereagents, protected amino acids and a recemization inhibitor (e.g. HOBtor HOOBt) may be directly added to the resin, or protected amino acidsmay be activated in advance in the form of symmetric acid anhydride,HOBt ester or HOOBt ester and, then, added to the resin.

The solvent used for the above-mentioned activation of protected aminoacids or the condensation thereof with a resin may be appropriatelyselected from those solvents known to be useful for polypeptide(protein) condensation reactions. Examples of useful solvents includeacid amides (e.g. N,N-dimethylformamide, N,N-dimethylacetamide, ofN-methylpyrrolidone), halogenated hydrocarbons (e.g. methylene chloride,or chloroform), alcohols (e.g. trifluoroethanol), sulfoxides (e.g.dimethyl sulfoxide), ethers (e.g. pyridine, dioxane, tetrahydrofuran),nitriles (e.g. acetonitrile or propionitrile), esters (e.g. methylacetate or ethyl acetate), and suitable mixtures of these solvents. Thereaction temperature may be appropriately selected from the range knownto be useful for polypeptide-forming reactions; usually, the temperatureis selected from the range from about −20° C. to about 50° C. Theactivated amino acid derivative is usually used in 1.5- to 4-foldexcess. When the condensation is found insufficient as a result of testusing the ninhydrin reaction, sufficient condensation can be achieved byrepeating reactions without removing protective groups. When sufficientcondensation cannot be achieved even by repeating reactions, unreactedamino acids may be acetylated with acetic anhydride or acetylimidazoleso that they do not affect subsequent reactions.

Examples of useful protective groups for the amino group of rawmaterials include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, and Fmoc.

The carboxyl group can be protected, for example, in the form of analkyl ester (e.g. straight-chain, branched, or cyclic alkyl esters suchas methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, and so on), aralkyl ester (e.g.benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, benzhydryl, andso on), phenacyl ester, benzyloxycarbonylhydrazide,t-butoxycarbonylhydrazide or tritylhydrazide.

The hydroxyl group of serine can be protected, for example, byesterification or etherification. Examples of suitable groups for thisesterification include lower (C₁₋₆) alkanoyl groups such as acetyl,aroyl groups such as benzoyl, and carbonic acid-derived groups such asbenzyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable forthe etherification include benzyl, tetrahydropyranyl and t-butyl.

Examples of protective groups for the phenolic hydroxyl group oftyrosine include Bzl, Cl₂-Bzl, 2-nitrobenzyl, BrZ, and t-butyl.

Examples of protective groups for the imidazole ring of histidineinclude Tos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP,benzyloxymethyl, Bum, Boc, Trt and Fmoc.

Examples of raw materials with activated carboxyl groups include thecorresponding acid anhydrides, azides and active esters (esters ofalcohols such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, p-nitrophenol, HONB,N-hydroxysuccinimide, N-hydroxyphthalimide and HOBt). Examples of rawmaterials with activated amino groups include the correspondingphosphoric acid amides.

Methods for removing (eliminating) protective groups include, forexample, catalytic reduction in a hydrogen stream in the presence of acatalyst such as Pd black or Pd-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid or mixtures thereof, base treatment withdiisopropylethylamine, triethylamine, piperidine, piperazine, or thelike, and reduction with sodium in liquid ammonia. The eliminationreaction by the above-mentioned acid treatment is generally conducted attemperatures of about −20° C. to about 40° C. In the acid treatment, itis effective to add a cation scavenger such as anisole, phenol,thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol or1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the protectivegroup for the imidazole of histidine is removed by thiophenol treatment.The formyl group used as the protective group for the indole oftryptophan may be removed by the above-mentioned deprotection by theacid treatment in the presence of 1,2-ethanedithiol, 1,4-butanedithiolor the like, or by alkali treatment using dilute sodium hydroxide,dilute ammonia or the like.

The protection of functional groups in raw materials that should not beinvolved in the reaction, protective groups therefor, the removal ofthese protective groups and the activation of functional groups involvedin the reaction can be appropriately selected from groups or methodspublicly known.

An alternative method for obtaining amides of a polypeptide of interestcomprises, for example, protecting the α-carboxyl group of theC-terminal amino acid by amidation, extending the peptide (polypeptide)chain to a desired length on the side of the amino group, preparing apolypeptide with its N-terminal α-amino group selectively deprotected,preparing a polypeptide with its C-terminal carboxyl group selectivelydeprotected, and condensing these two polypeptides in a mixed solventsuch as described above. Details of this condensation reaction are thesame as described above. After purification of the protected polypeptidethus obtained by condensation, all the protective groups are removed bythe method described above to thereby provide a crude polypeptide ofinterest. This crude polypeptide is purified by various knownpurification techniques and the major fractions are lyophilized toprovide the desired polypeptide in an amide form.

A method for obtaining esters of a polypeptide of interest comprises,for example, condensing the α-carboxyl group of the C-terminal aminoacid with a desired alcohol to prepare the corresponding amino acidester, and subjecting this ester to the same procedures as describedabove in the preparation of amides to thereby provide the desiredpolypeptide in an ester form.

The polypeptide of the invention or salt thereof can be produced bypublicly known methods for peptide synthesis. The method for peptidesynthesis may be solid-phase synthesis or liquid-phase synthesis.Briefly, a peptide of interest can be produced by condensing a partialpeptide or amino acid capable of constituting the partial peptide of theinvention with the residual part thereof and, if the product hasprotective groups, removing the protective groups. Examples ofcondensation methods and methods for removal of protective groupspublicly known include those described in the following references (1)to (5).

-   (1) M. Bodanszky & M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, 1966-   (2) Schroeder & Luebke, The Peptide, Academic Press, New York, 1965-   (3) Nobuo izumiya et al., Fundamentals and Experiments in Peptide    Synthesis, Maruzen, 1975-   (4) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment    Series 1, Polypeptide Chemistry IV, 205, 1977-   (5) Haruaki Yajima (ed.), Development of Drugs (Continued), Vol. 14,    Peptide Synthesis, Hirokawa Shoten

After the reaction, the polypeptide of the invention can be isolated andpurified by a combination of conventional purification techniques suchas solvent extraction, distillation, column chromatography, liquidchromatography, and recrystallization. When the polypeptide thusobtained is a free form, it can be converted to a suitable salt bypublicly known methods or methods based thereon. On the contrary, whenthe polypeptide is obtained in a salt form, it can be converted to afree form or another salt by publicly known methods or methods basedthereon.

The DNA encoding the polypeptide of the invention may be any DNA as longas it comprises a base sequence encoding the above-described polypeptideof the invention. The DNA may be genomic DNA, cDNA derived from theabove-mentioned cells or tissues, or synthetic DNA.

Vectors used for library may be any vectors such as bacteriophage,plasmid, cosmid, phagemid, and so on. Alternatively, total RNA or mRNAfraction may be prepared from the above-mentioned cells or tissues,followed by direct amplification by reverse transcriptase polymerasechain reaction (hereinafter, referred to as “RT-PCR”).

Examples of DNA encoding the polypeptide of the invention include a DNAcomprising the base sequence as shown in SEQ ID NO: 9 or SEQ ID NO: 22;or a DNA which has a base sequence hybridizing to the base sequence asshown in SEQ ID NO: 9 or SEQ ID NO: 22 under high stringent conditionsand encodes a polypeptide having a property (e.g. immunogenicity)substantially identical with the corresponding property of thepolypeptide of the invention. Any of such DNA may be used.

As a DNA hybridizable to the base sequence as shown in SEQ ID NO: 9under high stringent conditions, a DNA comprising a base sequence havingabout 60% or more, preferably about 70% or more, and more preferablyabout 80% or more homology to the base sequence as shown in SEQ ID NO: 9may be used, for example.

As a DNA hybridizable to the base sequence as shown in SEQ ID NO: 22under high stringent conditions, a DNA comprising a base sequence havingabout 60% or more, preferably about 70% or more, and more preferablyabout 80% or more homology to the base sequence as shown in SEQ ID NO:22 may be used, for example.

Hybridization can be carried out according to publicly known methods ormethods based thereon, e.g. those methods described in MolecularCloning, 2nd Ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press,1989) and the like. When commercial libraries are used, hybridizationcan be carried out in accordance with the methods described in theinstructions attached to the kits; preferably, hybridization is carriedout under high stringent conditions.

“High stringent conditions” refers to, for example, conditions wheresodium concentration is about 19-40 mM, preferably about 19-20 mM, andtemperature is about 50-70° C., preferably about 60-65° C.

As a DNA encoding the polypeptide of the invention having the amino acidsequence as shown in SEQ ID NO: 10, a DNA having the base sequence asshown in SEQ ID NO: 9 may be used, for example. As a DNA encoding thepolypeptide of the invention having the amino acid sequence as shown inSEQ ID NO: 19, a DNA having the base sequence as shown in SEQ ID NO: 22may be used, for example.

A DNA encoding the entire polypeptide of the invention can be clonedeither by PCR amplification using synthetic DNA primers each having apartial base sequence encoding the polypeptide of the invention, or byhybridization of DNA fragments inserted into a suitable vector to alabeled DNA fragment encoding a part or full length of the polypeptideof the invention or a labeled synthetic DNA fragment. The hybridizationcan be carried out, for example, according to the method described inMolecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring HarborLab. Press, 1989). When commercial libraries are used, the hybridizationcan be carried out according to the instructions attached thereto.

Substitution of the base sequence of a DNA can be performed by publiclyknown methods such as ODA-LA PCR, gapped duplex method, Kunkel methodand the like using publicly known kits such as Mutan™-Super Express Km(Takara), Mutan™-K (Takara), etc.

The cloned DNA encoding the polypeptide of the invention may be used asit is or after digestion with restriction enzymes or addition oflinkers, if desired. The DNA may have ATG at its 5′ end as thetranslation initiation codon and TAA, TGA, or TAG at its 3′ end as thetranslation termination codon. The translation initiation andtermination codons may be added by using appropriate synthetic DNAadapters.

Expression vectors for the polypeptide of the invention can be preparedby, for example, (a) cutting out a DNA fragment of interest from a DNAencoding the polypeptide of the invention and (b) ligating the DNAfragment to an appropriate expression vector downstream of its promoter.

Examples of vectors include plasmids derived from Escherichia coli (e.g.pBR322, pBR325, pUC12, and pUC13); plasmids derived from Bacillussubtilis (e.g. pUB110, pTP5 and pC194); plasmids derived from yeast(e.g. pSH19 and pSH15); bacteriophages such as λ-phage; animal virusessuch as retrovirus, vaccinia virus, baculovirus; and other vectors suchas pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and so on.

Any promoter may be used in the invention as long as it is appropriatefor the host that will be used for expressing a gene of interest. Whenthe host is an animal cell, examples of promoters include SRα promoter,SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter and β-actinpromoter.

Among these promoters, CMV (cytomegalovirus) promoter, SRα promoter orthe like is preferably used. When the host is Escherichia, trp promoter,lac promoter, recA promoter, λP_(L) promoter, Ipp promoter, T7 promoteror the like is preferably used. When the host is Bacillus, SPO1promoter, SPO2 promoter, penP promoter or the like is preferably used.When the host is a yeast, PHO5 promoter, PGK promoter, GAP promoter, ADHpromoter, or the like is preferably used. When the host is an insectcell, polyhedrin promoter, P10 promoter, or the like is preferably used.

The expression vectors may, if desired, further comprise enhancers,splicing signals, polyadenylation signals, selective markers, SV40replication origin (hereinafter, sometimes abbreviated to “SV40 ori”)and the like. Examples of selective markers include dihydrofolatereductase (hereinafter, sometimes abbreviated to “dhfr”) gene[methotorexate (MTX) resistance], ampicillin resistance gene(hereinafter, sometimes abbreviated to “Amp^(r)”), neomycin resistancegene (hereinafter, sometimes abbreviated to “Neo^(r)”: Geneticinresistance) and the like. When dhfr gene-deficient Chinese hamster cellsare used in combination with dhfr gene as a selective marker,recombinant cells may be selected even in a thymidine-free medium.

Furthermore, a signal sequence appropriate for the host may be added, ifnecessary, to the N-terminal of the polypeptide. When the host isEscherichia, the utilizable signal sequences may include PhoA signalsequence, OmpA signal sequence, or the like may be added. When the hostis Bacillus, α-amylase signal sequence, subtilisin signal sequence, orthe like may be added. When the host is yeast, MFα signal sequence, SUC2signal sequence, or the like may be added. When the host is an animalcell, insulin signal sequence, α-interferon signal sequence, antibodymolecule signal sequence, or the like may be added.

When the polypeptide of the invention has such a signal sequence asdescribed above, the polypeptide of the invention is secreted out ofcells effectively.

Transformants (or transfectants) can be prepared by using the thusconstructed vector comprising a DNA encoding the polypeptide of theinvention.

Examples of hosts include bacteria belonging to the genus Escherichia,bacteria belonging to the genus Bacillus, yeasts, insect cells, insects,and animal cells.

Specific examples of bacteria belonging to the genus Escherichia includeE. coli K12 DH1 (Proc. Natl. Acad. Sci. USA, Vol. 60, 160 (1968)), JM103(Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal ofMolecular Biology, Vol. 120, 517 (1978)), HB101 (Journal of molecularBiology, Vol, 41, 459 (1969)) and C600 (Genetics, Vol. 39, 440 (1954)).

Specific examples of bacteria belonging to the genus Bacillus include B.subtilis MI114 (Gene, Vol. 24, 255 (1983)) and 207-21 (Journal ofBiochemistry, Vol. 95, 76 (1984)).

Specific examples of yeasts include Saccharomyces cerevisiae AH22,AH22R⁻, NA87-11A, DKD-SD and 20B-12, Schizosaccharomyces pombe NCYC1913and NCYC2036, and Pichia pastoris KM71.

Specific examples of insect cells include, when the virus used is AcNPV,a cell line derived from larvae of Spodoptera frugiperda (Sf cells), MG1cells derived from the midgut of Trichoplusia ni, High Five™ cellsderived from eggs of Trichoplusia ni, Mamestra brassicae-derived cellsand Estigmena acrea-derived cells. When the virus used is BmNPV, insectcells such as a silkworm-derived cell line (Bombyx mori N cells; BmNcells) may be used. Specific examples of Sf cells useful in theinvention include Sf9 cells (ATCC CRL 1711) and Sf21 cells (bothdisclosed in Vaughn J. L. et al., In Vivo, 13, 213-217 (1977)).

Specific examples of insects include larvae of silkworm (Maeda et al.,Nature, 315, 592 (1985)).

Specific examples of animal cells include a simian cell COS-7 (COS7),Vero cells, a Chinese hamster cell CHO(CHO cells), a dhfr gene-deficientChinese hamster cell CHO (hereinafter, abbreviated to “CHO(dhfr⁻)cells”), mouse L cells, mouse AtT-20 cells, mouse myeloma cells, rat GH3cells, and human FL cells.

Transformation of bactera belonging to the genus Escherichia can beperformed in accordance with methods disclosed, for example, in Proc.Natl. Acad. Sci. USA, Vol. 69, 2110 (1972) and Gene, Vol. 17, 107(1982).

Transformation of bacteria belonging to the genus: Bacillus can beperformed in accordance with methods disclosed, for example, inMolecular & General Genetics, Vol. 168, 111 (1979).

Transformation of yeasts can be performed in accordance with methodsdisclosed, for example, in Methods in Enzymology, 194, 182-187 (1991)and Proc. Natl. Acad. Sci. USA, Vo. 75, 1929 (1978).

Transformation of insect cells or insects can be performed in accordancewith methods disclosed, for example, in Bio/Technology, 6, 47-55 (1988).

Transformation of animal cells can be performed by methods disclosed,for example, in Cell Engineering, Separate Vol. 8, New Cell EngineeringExperiment Protocol, 263-267 (1995) (Shujunsha Co.) and Virology, Vol.52, 456 (1973).

Thus, transformants transformed with an expression vector comprising aDNA encoding the polypeptide of the invention can be obtained.

As a medium to culture transformants obtained from Escherichia orBacillus bacteria as hosts, a liquid medium is appropriate. The mediumis allowed to contain carbon sources, nitrogen sources, minerals, and soon which are necessary for the growth of the transformant. As carbonsources, glucose, dextrin, soluble starch, sucrose or the like may beenumerated. As nitrogen sources, organic or inorganic substances such asammonium salts, nitrates, corn steep liquor, peptone, casein, yeastextract, meat extract, bean cake, potato extract, or the like may beenumerated. As minerals, calcium chloride, sodium dihydrogen phosphate,magnesium chloride, or the like may be enumerated. Further, yeastextract, vitamins, growth-promoting factors, etc. may also be added tothe medium. Preferable pH of the medium is about 5 to about 8.

As a medium to culture Escherichia bacteria, M9 medium containingglucose and casamino acid (Miller, Journal of Experiments in MolecularGenetics, 431-433, Cold Spring Harbor Laboratory, New York, (1972)) ispreferable, for example. If necessary, drugs such as 3β-indolyl acrylicacid can be added to the medium to improve efficiency of the promoter.

When the host is an Escherichia bacterium, the transformant is culturedusually at about 15-43° C. for about 3-24 hours. If necessary, aerationand stirring may be applied.

When the host is a Bacillus bacterium, the transformant is culturedusually at about 30-40° C. for about 6-24 hours. If necessary, aerationand stirring may also be applied.

As a medium to culture transformants obtained from yeasts as hosts, amedium such as Burkholder minimum medium (Bostian, K. L. et al., Proc.Natl. Acad. Sci. USA, Vol. 77, 4505 (1980)) or SD medium containing 0.5%casamino acid (Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, Vol.81, 5330 (1984)) may be used, for example. It is preferable that the pHof the medium is adjusted to about 5 to about 8. The transformant iscultured usually at about 20-35° C. for about 24-72 hours. If necessary,aeration and stirring may be applied.

As a medium to culture transformants obtained from insect cells orinsects as hosts, Grace's Insect Medium (Grace, T. C. C., Nature, 195,788 (1962)) supplemented with additives such as inactivated 10% bovineserum may be used, for example. It is preferable that the pH of themedium is adjusted to about 6.2-6.4. The transformant is culturedusually at about 27° C. for about 3-5 days. If necessary, aeration andstirring may be applied.

As a medium to culture transformants obtained from animal cells ashosts, examples of useful media include MEM medium (Science, Vol. 122,501 (1952)) containing about 5-20% fetal calf serum, DMEM medium(Virology, Vol. 8, 396 (1959)), RPMI 1640 medium (Journal of theAmerican Medical Association, Vol. 199, 519 (1967)), 199 medium(Proceedings of the Society of the Biological Medicine, Vol. 73, 1(1950)). Preferable pH of the medium is about 6 to about 8. Thetransformant is cultured usually at about 30-40° C. for about 15-60hours. If necessary, aeration and stirring may be applied.

Thus, the polypeptide of the invention can be produced inside oftransformant cells, in their cell membranes, or outside of these cells.

Separation and purification of the polypeptide from the resultantculture can be carried out, for example, according to the methodsdescribed below.

For extraction of the polypeptide of the invention from culturedmicroorganisms or cells, the microorganisms or cells are harvested bypublicly known methods after the cultivation, suspended in a suitablebuffer, and disrupted by sonication or by lysozyme and/or freezing andthawing, etc. Then, a crude extract of the polypeptide is obtained bycentrifugation or filtration. The buffer may contain a proteindenaturing agent such as urea or guanidine hydrochloride, or asurfactant such as Triton X-100™. If the polypeptide is secreted intothe culture broth, the supernatant is separated from the microorganismsor cells after completion of the cultivation and collected by publiclyknown methods.

Purification of the polypeptide contained in the thus obtained culturesupernatant or extract can be performed by an appropriate combination ofpublicly known methods for separation and purification. These publiclyknown methods include methods utilizing solubility such as salting outor sedimentation with solvents, methods mainly utilizing difference inmolecular weight such as dialysis, ultrafiltration, gel filtration andSDS-polyacrylamide gel electrophoresis, methods utilizing difference inelectric charge such as ion-exchange chromatography, methods utilizingspecific affinity such as affinity chromatography, methods utilizingdifference in the hydrophobicity such as reversed-phase high-performanceliquid chromatography, and methods utilizing difference in isoelectricpoint such as isoelectric electrophoresis.

When the thus obtained polypeptide is a free form, the polypeptide canbe converted into a salt by publicly known methods or methods basedthereon. On the contrary, when the polypeptide is obtained in a saltform, the salt can be converted into a free form or another saltaccording to publicly known methods or methods based thereon.

The polypeptide produced by the transformant can be arbitrarily modifiedor a part thereof can be removed therefrom by using an appropriateprotein modification enzyme or proteolytic enzyme before or after thepurification. Examples of such enzymes include trypsin, chymotrypsin,arginyl endopeptidase, protein kinase and glycosidase.

The presence of the polypeptide of the invention thus obtained can bemeasured by enzyme immunoassay, Western blot analysis or the like usingspecific antibodies.

Antibodies to the polypeptide of the invention or salt thereof may beeither polyclonal or monoclonal antibodies as long as they can recognizethe polypeptide of the invention or salt thereof.

Antibodies to the polypeptide of the invention or salt thereof can beprepared using the polypeptide of the invention as antigen and accordingto publicly known producing methods for antibody or anti-serumpreparation.

[Preparation of Monoclonal Antibodies]

(a) Preparation of Monoclonal Antibody-Producing Cells

The polypeptide of the invention or salt thereof is administered towarm-blooded animals either alone or together with a carrier or diluentto a site capable of producing antibodies upon the administration. Inorder to enhance the ability to produce antibodies, complete Freund'sadjuvants or incomplete Freund's adjuvants may also be administered. Theadministration is usually carried out once in every two to six weeks andtwo to ten times in the total. Examples of warm-blooded animals utilizedinclude monkey, rabbit, dog, guinea pig, mouse, rat, sheep, goat andchicken. Among them, mouse or rat is used preferably.

In the preparation of monoclonal antibody-producing cells, individualswith detectable antibody titers are selected from warm-blooded animals(e.g. mice) immunized with antigen. Then, the spleen or lymph nodes arecollected from them two to five days after the final immunization, andantibody-producing cells contained therein are fused with myeloma cellsof a homologous or heterologous animal to thereby obtain monoclonalantibody-producing hybridomas. Measurement of antibody titers inantisera may be carried out, for example, by reacting a labeledpolypeptide, which will be described later, with the antiserum followedby measuring the activity of the labeling agent bound to the antibody.The cell fusion may be carried out by a known method, for example, themethod of Koehler and Milstein (Nature, 256, 495, (1975)). Examples offusion promoters include polyethylene glycol (PEG), Sendai virus, etc.Preferably, PEG is used.

Examples of myeloma cells include myeloma cells of warm-blooded animalssuch as NS-1, P3U1, SP2/0, AP-1, etc. Preferably, P3U1 is used. Apreferable ratio of the number of antibody-producing cells used (spleencells) to the number of myeloma cells is about 1:1 to 20:1. When PEG(preferably, PEG 1000 to PEG 6000) is added at a concentration of about10-80% and the resultant cell mixture is incubated at 2040° C.(preferably, at 30-37° C.) for about one to ten minutes, an efficientcell fusion can be performed.

Various methods may be used for screening for monoclonalantibody-producing hybridomas. For example, hybridoma culturesupernatant is added to a solid phase (e.g. microplate) on which thepolypeptide antigen has been adsorbed either directly or with a carrier.Then, a radioactively or enzymatically labeled anti-immunoglobulinantibody (anti-mouse immunoglobulin antibody is used when mouse cellsare used in the cell fusion) or protein A is added thereto to detectmonoclonal antibodies bound to the solid phase. Alternatively, a methodmay be used in which hybridoma culture supernatant is added to a solidphase on which an anti-immunoglobulin antibody or protein A has beenadsorbed; then, a radioactively or enzymatically labeled polypeptide isadded thereto to thereby detect monoclonal antibodies bound to the solidphase.

Selection of monoclonal antibodies may be carried out by publicly knownmethods or methods based on them. Usually, selection can be carried outin a medium for culturing animal cells supplemented with HAT(hypoxanthine, aminopterin and thymidine). As a medium for selection andculturing, any medium may be used as long as hybridomas are capable ofgrowing therein. Examples of media include RPMI 1640 medium containingabout 1-20% (preferably about 10-20%) of fetal calf serum, GIT medium(Wako Pure Chemical Industries, Ltd.) containing about 1-20% of fetalcalf serum and a serum-free medium for hybridoma cultivation (SFM-101;Nissui Pharmaceutical Co.). The cultivation temperature is usually about20-40° C., preferably about 37° C. The cultivation period is usuallyfrom five days to three weeks, preferably one to two weeks. Thecultivation may be carried out usually under 5% carbon dioxide. Theantibody titer of hybridoma culture supernatant may be measured in thesame manner as in the above-mentioned measurement of the antibody titersin antisera.

(b) Purification of the Monoclonal Antibodies

Separation and purification of monoclonal antibodies may be carried outby publicly known methods, such as methods for separating/purifyingimmunoglobulin [e.g. salting-out, alcohol precipitation, isoelectricprecipitation, electrophoresis, adsorption/desorption using ionexchangers (e.g. DEAE), ultracentrifugation, gel filtration, specificpurification methods in which only an antibody is collected by means ofan antigen-binding solid phase or active adsorbent such as protein A orprotein G, followed by dissociation of the bond].

[Preparation of Polyclonal Antibodies]

The polyclonal antibody of the invention can be produced by publiclyknown methods or methods based on them. For example, an immunogen(antigen polypeptide) per se or a complex of the immunogen and a carrierprotein is prepared. Then, using the immunogen or the complex,warm-blooded animals are immunized in the same manner as described forthe production of monoclonal antibodies. Fractions containing theantibody against the polypeptide of the invention or salt thereof areharvested from the immunized animals, followed by separation andpurification of the antibody.

With respect to the immunogen-carrier protein conjugate for use in theimmunization of warm-blooded animals, the kind of carrier protein andthe mixing ratio of the carrier and the hapten are not particularlyrestricted as long as antibodies are produced efficiently against thehapten cross-linked to the carrier. For example, bovine serum albumin,bovine thyroglobulin, hemocyanine, or the like is coupled to the haptenat a weight ratio of about 0.1-20:1, preferably about 1-5:1.

A variety of condensing agents can be used for the coupling between thehapten and the carrier. For example, glutaraldehyde, carbodiimide,maleimide, or active ester reagents containing a thiol or dithiopyridylgroup may be used.

The condensation product is administered to a warm-blooded animal eitheralone or together with a carrier or diluent at a site capable ofproducing antibodies upon the administration. In order to enhance theantibody production ability, complete Freund's adjuvant or incompleteFreund's adjuvant may also be administered. Administration is carriedout generally once in about every 2-6 weeks and about 3-10 times in thetotal.

Polyclonal antibodies can be recovered from the blood, abdominal dropsyor other body fluid, preferably from the blood, of the warm-bloodedanimal immunized as described above.

Polyclonal antibody titers in antisera can be determined in the samemanner as described above for the determination of monoclonal antibodytiters in antisera. The separation and purification of polyclonalantibodies can be carried by the same methods for separation andpurification of immunoglobulin as those described for the separation andpurification of monoclonal antibodies.

With respect to the antisense DNA having a base sequence complementaryto or substantially complementary to a DNA encoding the polypeptide ofthe invention (hereinafter, sometimes referred to as the “DNA of theinvention”), any antisense DNA may be used as long as it has a basesequence complementary to or substantially complementary to the DNA ofthe invention and is able to inhibit the expression of the DNA.

A base sequence substantially complementary to the DNA of the inventionrefers to, for example, a base sequence having about 70% or more,preferably about 80% or more, more preferably about 90% or more, mostpreferably about 95% or more homology to the full-length or partial basesequence of the complementary base sequence to the DNA of the invention(i.e., the complementary strand to the DNA of the invention).Particularly preferable is an antisense DNA having about 70% or more,preferably about 80% or more, more preferably about 90% or more, mostpreferably about 95% or more homology to a partial base sequence of thecomplementary strand to the DNA of the invention that is complementaryto a base sequence encoding an N-terminal portion of the polypeptide ofthe invention (e.g. base sequence encoding a region adjacent to theinitiation codon). These antisense DNAs can be synthesized with DNAsynthesizers that are publicly known.

Hereinbelow, uses of the polypeptide of the invention, amide or esterthereof, or salt thereof (sometimes, they are simply referred to as the“polypeptide of the invention”); the DNA encoding the polypeptide of theinvention (sometimes referred to as the “DNA of the invention”); theantibody to the polypeptide of the invention, amide or ester thereof, orsalt thereof (sometimes referred to as the “antibody of the invention”);and the antisense DNA will be described.

(1) Therapeutic and/or Prophylactic for Various Diseases where Cubilinis Involved

Cubilin exists as a membrane protein in vivo and binds to apolipoproteinA-I. The polypeptide of the invention, which is a partial fragment ofcubilin having the ability to bind to apolipoprotein A-I, is able torepress the catabolism of apolipoprotein A-I by binding toapolipoprotein A-I contained in the blood. Therefore, the polypeptideand the DNA of the invention may be used as medicines such astherapeutics and/or prophylactics for diabetes, obesity,arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders. It ispossible to allow the role of the polypeptide of the invention tomanifest sufficiently in patients by, for example, (a) administering theDNA of the invention to patients and allowing the polypeptide of theinvention to express in vivo; (b) inserting the DNA of the inventioninto cells, allowing the expression of the polypeptide of the inventiontherein, and transplanting the cells into patients; or (c) administeringthe polypeptide of the invention to patients.

When the DNA of the invention is used as the above-mentioned therapeuticand/or prophylactic agent, the DNA per se or the DNA inserted into anappropriate vector such as a retrovirus vector, adenovirus vector,adeno-associated virus vector, etc. may be administered to human orother warm-blooded animals using conventional means. The DNA of theinvention or a vector into which the DNA of the invention is insertedmay be administered as it is or after formulation with physiologicallyacceptable carriers such as adjuvants to promote uptake. Usually, theDNA or the vector may be administered parenterally by means of, e.g., agene gun or a catheter such as hydrogel catheter.

When the polypeptide of the invention is used as the above-describedtherapeutic and/or prophylactic agent, at least 90%, preferably 95% ormore, more preferably 98% or more, still preferably 99% or more purifiedpolypeptide of the invention is used.

The polypeptide of the invention may be used, for example, orally in theform of tablets (sugar-coated, if necessary), capsules, elixirs,microcapsules or the like; or parenterally in the form of injectionssuch as aseptic solutions or suspensions in water or otherpharmaceutically acceptable liquids. These preparations may be produced,for example, by mixing the polypeptide of the invention withphysiologically acceptable carriers, flavoring agents, excipients,vehicles, antiseptics, stabilizers, binders, etc. in unit dosage formsrequired for preparing generally approved pharmaceutical preparations.The amounts of active ingredients in these formulations are decided sothat an appropriate dose within the specified range can be obtained.

Examples of additives miscible with tablets, capsules, etc. includebinders such as gelatin, corn starch, tragacanth and gum arabic,excipients such as crystalline cellulose, swelling agents such as cornstarch, gelatin and alginic acid, lubricants such as magnesium stearate,sweetening agents such as sucrose, lactose and saccharin, and flavoringagents such as peppermint, akamono oil and cherry. When the unit dosageform is capsule, liquid carrier such as oils and fats may further beincluded in addition to the above-mentioned materials. Sterilecompositions for injection can be formulated according to conventionalpractices in pharmaceutical manufacturing, e.g., by dissolving orsuspending active ingredients, naturally occurring vegetable oils suchas sesame oil, coconut oil, etc. in vehicles such as water forinjection.

Examples of aqueous liquids for injection include physiological salineand isotonic solutions containing glucose and other auxiliary agents(e.g. D-sorbitol, D-mannitol, sodium chloride, etc.). They may be usedin combination with a suitable auxiliary solubilizer such as alcohol(e.g. ethanol, etc.), polyalcohol (e.g. propylene glycol, polyethyleneglycol, etc.), nonionic surfactant (e.g. Polysorbate 80™, HCO-50, etc.).Examples of oily liquids for injection include sesame oil, soybean oil,etc. They may be used in combination with an auxiliary solubilizer suchas benzyl benzoate, benzyl alcohol, etc. In addition, buffers (e.g.phosphate buffer, sodium acetate buffer, etc.), soothing agents (e.g.benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g.human serum albumin, polyethylene glycol, etc.), preservatives (e.g.benzyl alcohol, phenol, etc.), antioxidants, etc. may also be admixedtherewith. Usually, the prepared injections are filled in appropriateampoules.

Since the thus obtained preparations are safe and of low toxicity, theycan be administered to human or other warm-blooded animals (e.g., rat,mouse, guinea pig, rabbit, avian, sheep, pig, bovine, horse, cat, dog,monkey, etc.).

Dose of the polypeptide of the invention may vary depending upon thetarget disease, the target to be administered, administration route, andso on. However, when the polypeptide of the invention is administeredorally for treating diabetes, obesity, arteriosclerosis, hyperlipemia,hypertriglyceridemia, hypo-high density lipoproteinemia,hypoapolipoproteinemia A-I or nervous disorders, generally thepolypeptide of the invention is administered to adult (60 kg in bodyweight) at a dose of about 1-1000 mg/day, preferably about 10-500mg/day, more preferably about 10-200 mg/day. With respect to parenteraladministration, when the polypeptide of the invention is administered toadult (60 kg in body weight) in the form of an injection for treatingdiabetes, obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia,hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I or nervousdisorders, it is convenient to inject the polypeptide of the inventioninto the affected part of the body at a dose of about 1-1000 mg/day,preferably about 1-200 mg/day, and more preferably about 10-100 mg/day,though the dose per administration may vary depending on the patient tobe treated, the target disease, etc. For other animals, correspondingdoses may be administered after conversion of the above-mentioned valuesper 60 kg based on actual body weights.

(2) Screening for Candidate Compounds for Medicine to Diseases

(i) When the expression level of cubilin is abnormally enhanced, variousdiseases such as, for example, hyperlipemia, hypertriglyceridemia,hypo-high density lipoproteinemia, hypoapolipoproteinemia A-I, aftermeal hyperlipemia, diabetes, obesity, arteriosclerosis, myocardialinfarction or angina occur.

Therefore, compounds or salts thereof that inhibit the binding ofcubilin to apolipoprotein A-I may be used as medicines such astherapeutics and/or prophylactics for, e.g., hyperlipemia,hypertriglyceridemia, hypo-high density lipoproteinemia,hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity,arteriosclerosis, myocardial infarction or angina.

(ii) On the other hand, since cubilin exists as a membrane protein invivo and binds to apolipoprotein A-I, various diseases such as renaldisorders, nephritis, nephropathy, proteinuria, nervous disorders andvitamin B₁₂ deficiency occur when cubilin or DNA encoding the same isabnormal or deficient, or cubilin expression is abnormally reduced.

Therefore, compounds or salts thereof that promote the binding ofcubilin to apolipoprotein A-I may be used as medicines such astherapeutics and/or prophylactics for various diseases such as renaldisorders, nephritis, nephropathy, proteinuria, neurological disordersand vitamin B₁₂ deficiency.

Therefore, use of cubilin for screening for those compounds or saltsthereof that inhibit or promote the function of cubilin can becontemplated.

However, since cubilin is a large protein consisting of more than 3,500amino acids, non-specific adsorption or binding, or degradation ofcubilin molecules per se may occur when cubilin molecules are used inthe screening for such compounds or salts thereof; due to thesedrawbacks, it is impossible to carry out highly sensitive and efficientscreening.

On the other hand, the screening method using the polypeptide of theinvention does not have such drawbacks. By using this screening method,compounds or salts thereof that inhibit or promote the binding betweenthe polypeptide of the invention and apo A-I can be screened with highsensitivity and efficiently. Since such compounds or salts thereofinhibit or promote the binding between cubilin and apo A-I, they can beused as medicines such as therapeutics and/or prophylactics for theabove-mentioned various diseases.

That is, the present invention provides:

-   1) {circle over (1)} a method for screening for compounds or salts    thereof that inhibit the binding between cubilin and apo A-I    (sometimes abbreviated to “inhibitor(s)” in “(2) Screening for    Candidate Compounds for Medicine to Treat Diseases”) or compounds or    salts thereof that promote the binding between cubilin and apo A-I    (sometimes abbreviated to “promoter(s)” in “(2) Screening for    Candidate Compounds for Medicine to Treat Diseases”), wherein the    method is characterized by using the polypeptide of the invention;    and {circle over (2)} a screening kit for inhibitors or promoters    comprising the polypeptide of the invention (sometimes referred to    as the “screening kit of the invention” in “(2) Screening for    Candidate Compounds for Medicine to Treat Diseases”). More    specifically, the present invention provides:-   2) {circle over (1)} a method for screening for promoters or    inhibitors, comprising comparing (i) the case where apo A-I is    contacted with the polypeptide of the invention and (ii) the case    where apo A-I and a test compound are contacted with the polypeptide    of the invention; and {circle over (2)} a screening kit for    promoters and inhibitors, comprising the polypeptide of the    invention and apo A-I.

Specifically, the above-mentioned screening method is characterized bymeasuring and comparing the amounts of binding of apo A-I to thepolypeptide of the invention or the amounts of binding of thepolypeptide of the invention to apo A-I in the cases (i) and (ii).

These amounts of binding can be measured by publicly known methods ormethods based on them.

Examples of test compounds include, but not limited to, peptides,proteins, non-peptidic compounds, synthetic compounds, fermentationproducts, cell extracts, plant extracts, and animal tissue extracts.These compounds may be either novel compounds or publicly knowncompounds.

As the peptide, partial peptides of apo A-I are preferable.

For example, a test compound that reduces the amount of binding in case(ii) above by about 20% or more, preferably 30% or more, more preferablyabout 50% or more compared to the amount of binding in case (i) abovemay be selected as a compound that inhibits the binding between cubilinand apo A-I. On the other hand, a test compound that increases theamount of binding in case (ii) above by about 20% or more, preferably30% or more, more preferably about 50% or more compared to the amount ofbinding in case (i) above may be selected as a compound that promotesthe binding between cubilin and apo A-I.

The compounds of salts thereof obtained by using the screening method orthe screening kit of the invention are compounds that are selected fromthe above-mentioned test compounds, e.g. peptides, proteins,non-peptidic compounds, synthetic compounds, fermentation products, cellextracts, plant extracts, animal tissue extracts, plasma, etc., and thatpromote or inhibit the function of cubilin (e.g. binding toapolipoprotein A-I).

As salts of such compounds, salts same as those salts of the polypeptideof the invention described herein earlier may be used.

Hereinbelow, the screening method will be described specifically.

The screening may be performed by SPA (scintillation proximity assay)method (Anal. Biochem. 1987 March; 161 (2): 494-500), fluorescencebinding assay, a method using a surface plasmon sensor, or a methodbased on them.

[I] SPA method may be performed as follows.

The polypeptide of the invention is bound to SPA beads directly orindirectly and then mixed with (i) apo A-I labeled with a radioactiveisotope or (ii) apo A-I labeled with a radioactive isotope and a testcompound. Subsequently, the fluorescence intensities in case (i) andcase (ii) are measured and compared.

More specifically, the method is performed as follows:

{circle over (1)} The polypeptide of the invention is bound to SPAbeads. The binding may be either direct or indirect

As a method for direct binding, the method described in the protocolattached to SPA beads may be used. As a method for indirect binding, thefollowing methods may be enumerated, for example:

-   -   (i) A method in which SPA beads coated with copper are used, and        histidine tag-added polypeptide of the invention are bound to        these SPA beads,    -   (ii) A method in which SPA beads coated with glutathione are        used, and a fusion protein composed of GST and the polypeptide        of the invention is bound to these SPA beads,    -   (iii) A method in which SPA beads coated with a secondary        antibody are used, and the polypeptide of the invention and        these SPA beads are bound via a linker that is an antibody to        the polypeptide,    -   (iv) A method in which SPA beads coated with a secondary        antibody are used, and the polypeptide of the invention to which        a tag such as FLAG or myc has been added is bound to these SPA        beads via an antibody to the tag,    -   (v) A method in which SPA beads coated with streptavidin are        used, and biotin-labeled polypeptide of the invention is bound        to these SPA beads,    -   (vi) A method in which SPA beads coated with streptavidin are        used, and the polypeptide of the invention to which a tag such        as FLAG or myc has been added is bound to these SPA beads via a        linker that is a biotin-labeled antibody to the tag.

When the polypeptide of the invention has sugar chains, the followingmethod may also be used:

-   -   (vii) A method in which SPA beads coated with WGA (wheat germ        agglutinin) are used, and the polypeptide of the invention is        bound to these SPA beads.

SPA beads coated with such materials may be prepared by publicly knownmethods or methods based on them. However, it would be convenient topurchase them from Amersham Pharmacia Biotech.

{circle over (2)} (i) When a tag is added to the polypeptide of theinvention, any conventional tag used in the art may be used as long asit can be detected specifically.

Preferable examples include tags consisting of several amino acids, suchas FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) or myc(Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu), and histidine tag that do notaffect the binding between the polypeptide of the invention and apo A-I.

By expressing a fusion composed of such a tag and the polypeptide of theinvention, a tag-added polypeptide of the invention can be obtained. Thefusion can be obtained by inserting a DNA encoding the polypeptide ofthe invention into a vector such as pFLAG-CMV-1, pSecTag orpcDNA3.1/His, introducing the vector into an appropriate host cell suchas E. coli, COS7 or CHO, and allowing the host cell to produce thepolypeptide in cells or cell membranes or outside of cells. The tag maybe added to any site of the polypeptide of the invention, but a sitethat does not affect the binding with apo A-I is preferable.Hereinafter, the polypeptide of the invention to which a tag has beenadded is sometimes referred to as “Tag-CUBIII”.

(ii) A fusion protein composed of GST and the polypeptide of theinvention can be prepared by publicly known methods or methods based onthem. The site of fusion may be any site of the polypeptide of theinvention, but a site that does not affect the binding with apo A-I ispreferable.

(iii) When apo A-I is labeled with a radioactive isotope (e.g. [¹²⁵I],[¹³¹I], [³H], [¹⁴C], [³⁵S] or [³³P]), the labeling may be performed bypublicly known methods or methods based on them. Specifically, apo A-Iis dissolved in 40 ml of 0.4 M glycine-NaOH buffer (pH 8.5) at 50 μg/ml,followed by addition of 1 mCi of ¹²⁵I for labeling reaction. To thereaction solution, 50 μl of Cloramin T solution (4 mg/ml) is added andmaintained at room temperature for 15 min. Then, 100 μl of metabisulfitesolution (16 mg/ml) is added thereto and stirred for 10 min. Theresultant reaction solution is extracted with 40 ml of acetone/diethylether (3:1, v/v). The resultant precipitate is dried, dissolved in aphosphate buffer and subjected to dialysis against the buffer to therebyobtain ¹²⁵I-labeled apo A-I.

(iv) When an anti-tag antibody is modified with biotin, an anti-tagantibody may be prepared by publicly known methods or methods based onthem using a tag such as FLAG or myc as an antigen. Such an antibody maybe either a monoclonal antibody or a polyclonal antibody. Commercialanti-tag antibodies may also be used. As an anti-myc antibody, Clone9E10 (Sigma) or the like may be used. As an anti-FLAG antibody, Clone M1(Sigma) or the like may be used.

The modification of anti-tag antibodies and the polypeptide of theinvention with biotin may be performed by publicly known methods ormethods based on them. Specifically, an anti-tag antibody or thepolypeptide of the invention is dissolved in a carbonate buffer (0.1 Msodium hydrogencarbonate, 0.1 M sodium chloride, pH 8.3) to give aconcentration of 1 mg/ml. To this solution, 50 μl of an aqueous solutionof Sulfo-NHS-Biotin (Pierce) (1 mg/ml) is added and mixed gentlyovernight at 4° C. Then, the reaction solution is dialyzed against 20 mMTris-HCl buffer (pH 7.4) containing 150 mM sodium chloride to performbiotin modification.

(v) After the procedures described in (i) to (iv) have been completed,the fluorescence intensity is measured with a scintillator. If thefluorescence intensity is decreased by the addition of a test compound,the compound can be selected as an inhibitor. If the fluorescenceintensity is increased by the addition of a test compound, the compoundcan be selected as a promoter.

Hereinbelow, operational procedures will be described more specifically.Briefly, 1 mg of streptavidin-yttrium silicate type SPA beads, 0.5 μl ofbiotin-modified myc monoclonal antibody (9E10; Sigma), 10-200 ng ofpurified polypeptide of the invention (CUBIII-myc-His fragment), 250,000cmp of radioactive iodine-labeled apo A-I, and 10 pmol to 100 pmol ofunlabeled apo A-I are mixed in 20 mM Tris-HCl buffer (pH 7.4) containing150 mM sodium chloride, 1.0 mM calcium chloride and 0.1% sodium azidehereinafter, referred to as the “binding reaction buffer”) to give areaction solution of 200 μl.

This reaction solution is mixed gently at 25° for 1 hr and centrifugedin a bench type centrifuge at 1,000 rpm for 2 min to precipitate SPAbeads. Then, the fluorescence emitted by SPA beads is measured with ascintillation counter. For the purpose of evaluation of substances thatinhibit or promote binding, a test substance is dissolved in theabove-mentioned binding reaction buffer and included in a 200 μlreaction solution. Substances that change scintillation counts areevaluated taking the scintillation count in the absence of the testsubstance as 100%.

The screening kit of the invention comprises labeled apo A-I, variouslycoated SPA beads and the polypeptide of the invention.

[II] Fluorescence apo A-I binding assay is performed using a platecoated with the fragment peptide of the invention.

Specifically, the assay is performed as described below. After thepolypeptide of the invention is solidified in a plate, labeled apo A-Iand a test compound are mixed and incubated on the plate. After washing,the amount of labeled apo A-I bound to the polypeptide is determined byan appropriate method.

For example, the polypeptide of the invention is diluted to 0.1-10 μg/mlin a buffer and solidified by incubation in a high binding ability-typemicroplate for fluorescence measurement (e.g. Black Clini plate enhancedbinding; Labsystems). As the buffer, a buffer containing 20 mM Tris-HCl(pH 7.4), 500 mM NaCl, 2 mM CaCl₂ and 0.1% sodium azide may be used, forexample.

After the polypeptide is solidified by an overnight or longerincubation, non-specific binding is inhibited with an appropriateblocking reagent. For this purpose, SuperBlock TBS (Pierce) may be used,for example.

After this blocking, the microplate is washed with a buffer to be usedin the subsequent binding reaction. Then, a mixture of labeled apo A-Iand a test compound is incubated on wells of the microplate to carry outcompetitive binding reaction. As the buffer, a buffer containing 20 mMTris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 0.1% sodium azide and 5%Block-Ace (Dainippon Pharmaceutical) may be used, for example. Forlabeling apo A-I particles, Sulfo-NHS-LC-LC-Biotin may be used, forexample. Specifically, apo A-I is dissolved in 50 mM sodium bicarbonate(pH 8.5) at a concentration of 1 mg/ml. To this solution, 1/10 volume ofSulfo-NHS-LC-LC-Biotin dissolved in pure water at 1 mg/ml is added andincubated overnight at 4° to thereby prepare biotin-labeled apo A-I. Asthe concentration of biotin-labeled apo A-I in the competitive bindingreaction solution, a concentration of approximately 0.1 μg/ml may beused, for example.

For quantitative determination of biotin-labeled apo A-I bound to thepolypeptide after washing, β-galactosidase-labeled streptavidin may beused, for example. For measuring its activity,4-methylumbelliferyl-β-D-galactopyranoside or the like may be used. Inthis case, the amount of biotin-labeled apo A-I specifically bound tothe polypeptide of the invention can be calculated and determined bymeasuring excitation light at around 365 nm and fluorescence emission ataround 460 nm.

[III] The method using a surface plasmon sensor may be performed usingBIACORE™ 3000 (Biacore) or the like according to the protocol attachedthereto. Specifically, this method may be performed as described below.

The polypeptide of the invention is bound to a sensor chip directly orindirectly, and

-   -   (i) apo A-I solution is applied to the sensor chip; or (ii) apo        A-I solution containing a test substance is applied to the        sensor chip. Then, the surface plasmon values in the cases        of (i) and (ii) are measured and compared.

If the fluorescence intensity is decreased by the addition of a testcompound, the compound can be selected as an inhibitor. If thefluorescence intensity is increased by the addition of a test compound,the compound can be selected as a promoter.

More specifically:

{circle over (1)} The polypeptide of the invention is fixed on thesensor chip. This fixing may be either direct fixing or indirect fixing.

Direct fixing may be performed by any binding method. Preferably, thepolypeptide is fixed on the sensor chip by covalent bond as NHS-ester.As a method for indirect fixing, a method based on the above-mentionedmethod may be used.

Alternatively, apo A-I may be fixed on a sensor chip, and a solution ofthe polypeptide of the invention may be applied to the sensor chip.

{circle over (2)} Subsequently, apo A-I solution is applied to thesensor chip.

{circle over (3)} The surface plasmon is measured.

Specifically, a sensor chip CM5 is treated with a 1:1 mixture of 0.2 Mn-ethyl-N′-(3 diethylaminopropyl)-carbodiimide aqueous solution and 0.05M N-hydroxysuccinimide aqueous solution. To this sensor chip, thepolypeptide of the invention (CUBIII-myc-His fragment) dissolved in 10mM sodium acetate (pH 4.0) at 40 μg/ml is applied to thereby fix thepolypeptide of the invention to censor chip CM5. For blocking, 1 Methanol amine (pH 8.5) is applied. As a buffer for measuring binding, abuffer containing 10 mM sodium dihydrogen phosphate, 150 mM sodiumchloride, 1.0 mM calcium chloride and 0.1% sodium azide (pH 7.4) isused. After equilibration with this buffer and stabilization of plasmonchanges, apo A-I diluted with this buffer to 50-500 nM is applied to thesensor chip to measure changes in surface plasmon. For the purpose ofevaluation of substances that inhibit or promote the binding, a testsubstance is dissolved in the above-described buffer and mixed with theapo A-I solution. By comparing changes in surface plasmon in the absenceof a test compound with changes in the presence of the test compound,binding inhibitors or promoters are evaluated.

The screening kit of the invention comprises a sensor chip on which thepolypeptide of the invention or apo A-I is fixed, and apo A-I or thepolypeptide.

Compounds or salts thereof that are obtainable by using the screeningmethod or screening kit of the invention are those compounds that areselected from, for example, peptides, proteins, non-peptidic compounds,synthetic compounds, fermentation products, cell extracts, plantextracts, animal tissue extracts and plasma, and that promote or inhibitthe function of the polypeptide of the invention.

As salts of such compounds, salts same as those salts of the polypeptideof the invention described herein earlier may be used.

When the compound obtained by using the screening method or screeningkit of the invention is used as the above-mentioned therapeutic and/orprophylactic agent, it may be used according to conventional procedures.For example, the compound may be formulated into tablets, capsules,elixirs, microcapsules, aseptic solutions, suspensions, etc. in the samemanner as described in the preparation of medicines comprising thepolypeptide of the invention.

Since the thus obtained pharmaceutical preparations are safe and of lowtoxicity, they can be administered to human or other warm-bloodedanimals (e.g., mouse, rat, rabbit, sheep, pig, bovine, horse, avian,cat, dog, monkey, etc.).

Dose levels of the compound or salt thereof may vary depending upon thetarget disease, the target to be administered, administration route, andso on. However, when a compound that promotes the binding between thepolypeptide of the invention and apo A-I is administered orally fortreating nephritis, nephropathy, hyper-high density lipoproteinemia,hyperapolipoproteinemia A-I or proteinuria, generally the compound isadministered to adult (60 kg in body weight) at a dose of about 0.1-100mg/day, preferably about 1.0-50 mg/day, more preferably about 1.0-20mg/day. With respect to parenteral administration, when a compound thatpromotes the binding between the polypeptide of the invention and apoA-I is administered to adult (60 kg in body weight) in the form of aninjection for treating nephritis, nephropathy, hyper-high densitylipoproteinemia, hyperapolipoproteinemia A-I or proteinuria, it isconvenient to inject the compound intravenously at a dose of about0.01-30 mg/day, preferably about 0.1-20 mg/day, and more preferablyabout 0.1-10 mg/day, though the dose per administration may varydepending on the target to be administered, the target disease, etc. Forother animals, corresponding doses may be administered after conversionof the above-mentioned values per 60 kg based on actual body weights.

On the other hand, when a compound that inhibits the binding between thepolypeptide of the invention and apo A-I is administered orally fortreating diseases such as hyperlipemia, hypertriglyceridemia, hypo-highdensity lipoproteinemia, hypoapolipoproteinemia A-I, after mealhyperlipemia, arteriosclerosis, myocardial infarction or angina,generally the compound is administered to adult (60 kg in body weight)at a dose of about 0.1-100 mg/day, preferably about 1.0-50 mg/day, morepreferably about 1.0-20 mg/day. With respect to parenteraladministration, when a compound that inhibits the binding between thepolypeptide of the invention and apo A-I is administered to adult (60 kgin body weight) in the form of an injection for treating diseases suchas hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia,arteriosclerosis, myocardial infarction or angina, it is convenient toinject the compound intravenously at a dose of about 0.01-30 mg/day,preferably about 0.1-20 mg/day, and more preferably about 0.1-10 mg/day,though the dose per administration may vary depending on the target tobe administered, the target disease, etc. For other animals,corresponding doses may be administered after conversion of theabove-mentioned values per 60 kg based on actual body weights.

(3) Quantitative Determination of the Polypeptide of the Invention orSalt Thereof

Since an antibody to the polypeptide of the invention (hereinafter,sometimes referred to as the “antibody of the invention”) canspecifically recognize the polypeptide of the invention and cubilin(hereinafter, sometimes referred to as the “polypeptide of theinvention” in “(4) Quantitative Determination of the Polypeptide of theInvention of Salt Thereof”), the antibody may be used for quantitativedetermination of the polypeptide of the invention contained in a samplesolution. In particular, the antibody may be used in quantitativedetermination by sandwich immunoassay technique.

The present invention provides:

-   (i) a method of quantitative determination of the polypeptide of the    invention in a sample solution, comprising reacting the antibody of    the invention with the sample solution and the polypeptide of the    invention labeled, competitively and determining the ratio of the    labeled polypeptide of the invention bound to the antibody; and (ii)    a method of quantitative determination of the polypeptide of the    invention in a sample solution, comprising reacting the sample    solution with the antibody of the invention insolubilized on a    carrier and another antibody of the invention labeled,    simultaneously or in succession and determining the activity of the    label on the insolubilized carrier.

A monoclonal antibody to the polypeptide of the invention (hereinafter,sometimes referred to as the “monoclonal antibody of the invention”) maybe used to quantitatively determine the polypeptide of the invention ormay be used for detection of the polypeptide by tissue staining. Forthese purposes, either antibody molecules per se or the F(ab′)₂, Fab′ orFab fragment thereof may be used.

Methods of quantitative determination of the polypeptide of theinvention using the antibody of the invention are not particularlylimited. Any measuring method may be used in which the amount ofantibody, antigen or antibody-antigen complex corresponding to theamount of the antigen in a sample solution (e.g. the amount of thepolypeptide of the invention) is detected by chemical of physical means,and then calculated from a standard curve prepared with a standardsolution containing a known amount of the antigen. For example,nephrometry, competitive method, immunometric method and sandwich methodmay be used suitably and, in terms of sensitivity and specificity, thesandwich method described later is particularly preferred.

Examples of labeling agents used in measuring methods utilizing labelingsubstances include radioisotopes, enzymes, fluorescent substances, andluminescent substances. Examples of radioisotopes include [¹²⁵I],[¹³¹I], [³] and [¹⁴C]. Preferred examples of enzymes are those which arestable and with high specific activity, e.g., β-galactosidase,β-glucosidase, alkali phosphatase, peroxidase and malate dehydrogenase.Examples of fluorescent substances include fluorescamine and fluoresceinisothiocyanate. Examples of luminescent substances include luminol,luminol derivatives, luciferin, and lucigenin. Further, a biotin-avidinsystem may also be used for binding an antibody or antigen with alabeling agent

Insolubilization of antigens or antibodies may be performed by physicaladsorption or by chemical binding usually used for insolubilizing orimmobilizing polypeptides or enzymes. Examples of carriers includeinsoluble polysaccharides such as agarose, dextran and cellulose;synthetic resins such as polystyrene, polyacrylamide and silicone; andglass.

In sandwich method, a sample solution is reacted with an insolubilizedmonoclonal antibody of the invention (primary reaction); then, anothermonoclonal antibody of the invention that is labeled is reactedtherewith (secondary reaction); and the activity of the labeling agenton the insolubilized carrier is measured to thereby quantitativelydetermine the amount of the polypeptide of the invention in the samplesolution. The primary reaction and the secondary reaction may beconducted in a reverse order, or they may be conducted simultaneously orwith an interval. The type of the labeling agent and the method ofinsolubilization may be the same as those described herein earlier. Inimmunoassays using the sandwich technique, the antibody insolubilized ona solid phase or the antibody labeled is not necessarily a singleantibody; a mixture of two or more antibodies may be used for thepurposes of enhancing the sensitivity of measurement, etc.

In the method of measuring the polypeptide of the invention by thesandwich method of the invention, the monoclonal antibodies of theinvention used in the primary and the secondary reactions are preferablythose antibodies wherein their sites binding of the polypeptide of theinvention are different from each other. For example, if the antibodyused in the secondary reaction recognizes the C-terminal region of thepolypeptide of the invention, an antibody that recognizes a site otherthan the C-terminal region, e.g. an N-terminal region, is preferablyused in the primary reaction.

The monoclonal antibody of the invention may be used in a measuringsystem other than the sandwich method, such as competitive methods,immunometric methods and naphrometry.

In competitive methods, an antigen in a sample solution and a labeledantigen are reacted competitively with an antibody; then, unreactedlabeled antigen (F) and labeled antigen bound to the antibody (B) areseparated (i.e. B/F separation); and the amount of the label of B or Fto thereby quantitatively determine the amount of the antigen in thesample solution. With respect to this reaction method, there are aliquid phase method in which a soluble antibody is used and the B/Fseparation is conducted with polyethylene glycol and a second antibodyto the above-mentioned antibody; and a solid phase method in which asolidified antibody is used as the first antibody or a soluble antibodyis used as the first antibody while a solidified antibody is used as thesecond antibody.

In immunometric methods, an antigen in a sample solution and asolidified antigen are reacted competitively with a specific amount of alabeled antibody, followed by separation of the solid phase from theliquid phase; or an antigen in a sample solution is reacted with anexcessive amount of a labeled antibody, and then a solidified antigen isadded to bind unreacted labeled antibody to the solid phase, followed byseparation of the solid phase from the liquid phase. Subsequently, theamount of label in one of the phases is measured to determine the amountof the antigen in the sample solution.

In nephrometry, the amount of insoluble precipitate generated as aresult of antigen-antibody reaction in a gel or solution is measured.Even when the amount of the antigen in a sample solution is small andthus only a small amount of such precipitate is obtained, lasernephrometry utilizing the scattering of laser can be used suitably.

In applying each of those immunological measuring methods to themeasuring method of the present invention, no special conditions oroperations are required. A measuring system for the polypeptide of thepresent invention may be constructed using the conventional conditionsand operational procedures in the relevant measuring method while takinginto account usual technical consideration of those skilled in the art.For details of these commonly used technical means, a variety ofreviews, reference books, etc. may be referred to.

For example, Hiroshi Irie (ed.): “Radioimmunoassay” (Kodansha, 1974);Hiroshi Irie (ed.): “Radioimmunoassay; Second Series” (Kodansha, 1979);Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Igaku Shoin, Japan,1978); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Second Edition)(Igaku Shoin, 1982); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay”(Third Edition) (Igaku Shoin, 1987); “Methods in Enzymology”, Vol. 70(Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (Academic Press) and the like may be referred to.

By using the antibody of the invention as described above, thepolypeptide of the invention can be quantitatively determined with highsensitivity.

Further, by quantitatively determining the concentration of thepolypeptide of the invention using the antibody of the invention, it ispossible to diagnose that a subject has a disease such as, e.g.,hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia,hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis,myocardial infarction or angina, or that a subject is very likely todevelop such a disease in the future, when an increase of decrease isdetected in the concentration of the polypeptide of the invention in thesubject.

Further, the antibody of the invention may be used for detecting thepolypeptide of the invention present in body fluid, tissue or othersamples. The antibody of the invention may also be used in thepreparation of antibody columns for use in the purification of thepolypeptide of the invention; in the detection of the polypeptide of theinvention in individual fractions generated in the course ofpurification; and in the analysis of the behavior of the polypeptide ofthe invention in test cells.

(4) Gene Diagnostic Agent

When used as a probe, the DNA of the invention can detect abnormalities(gene abnormalities) in DNA or mRNA encoding the polypeptide of theinvention in human or other warm-blooded animals (e.g. rat, mouse,guinea pig, rabbit, avian, sheep, pig, bovine, horse, cat, dog, monkey,etc.). Thus, the DNA of the invention is useful as a gene diagnosticagent for, e.g., damage, mutations or reduced expression of the DNA ormRNA encoding the polypeptide of the invention, or increase or excessiveexpression of the DNA or mRNA.

Gene diagnosis using the DNA of the invention described above, may beperformed by publicly known methods such as Northern hybridization orPCR-SSCP method (Genomics, Vol. 5, 874-879 (1989); Proc. Natl. Acad.Sci. USA 86: 2766-2770 (1989)).

When a reduction in expression is detected by Northern hybridization orwhen mutations are detected in the DNA by PCR-SSCP method, for example,it is possible to diagnose that the relevant subject is very likely tohave a disease such as renal disorders, nephritis, nephropathy,proteinuria, nervous disorders, or vitamin B₁₂ deficiency.

On the other hand, when excessive expression is detected by Northernhybridization, it is possible to diagnose that the relevant subject hasa disease such as, e.g., hyperlipemia, hypertriglyceridemia, hypo-highdensity lipoproteinemia, hypoapolipoproteinemia A-I, after mealhyperlipemia, arteriosclerosis, myocardial infarction or angina.

(5) Medicines Containing the Antibody of the Invention

The antibody of the invention that neutralizes the activity of thepolypeptide of the invention may be used as a therapeutic and/orprophylactic agent for, e.g., diseases caused by excessive expression ofthe polypeptide of the invention.

The neutralizing antibody of the invention specifically recognizes theapo A-I-binding site in cubilin or regions in the vicinity thereof.Therefore, like the inhibitor, the antibody may be used as medicinessuch as therapeutics and/or prophylactics for diseases such as, e.g.,hyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemiahypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis,myocardial infarction or angina.

For these purposes, either antibody molecules per se or the F(ab′)₂,Fab′ or Fab fragment thereof may be used.

Therapeutics and/or prophylactics for the above-mentioned diseasescomprising the antibody of the invention may be administered orally orparenterally to human or other warm-blooded animals (e.g., rat, rabbit,sheep, pig, bovine, cat, dog, monkey, etc.) in the form of a liquidpreparation without any processing or in appropriate forms ofpharmaceutical compositions. Dose levels may vary depending upon thepatient to be treated, the target disease, symptoms, administrationroute, and so on. However, for the purpose of treating diseases such ashyperlipemia, hypertriglyceridemia, hypo-high density lipoproteinemia,hypoapolipoproteinemia A-I, after meal hyperlipemia, arteriosclerosis,myocardial infarction or angina, it is convenient to inject the antibodyof the invention intravenously at a dose of about 0.01-20 mg/kg bodyweight, preferably about 0.1-10 mg/kg body weight, more preferably about0.1-5 mg/kg body weight per administration about one to five times aday, preferably about one to three times a day. In other parenteraladministration and oral administration, similar dose levels may be used.If symptoms are particularly heavy, the dose may be increasedaccordingly.

The antibody of the invention may be adminstered per se or in the formsof appropriate pharmaceutical compositions. The pharmaceuticalcompositions for the above administration comprise the antibody or saltthereof, pharmacologically acceptable carriers, and diluents orexcipients. Such compositions are provided in forms appropriate for oralor parenteral administration.

For example, compositions for oral administration include solid orliquid preparations such as tablets (including sugar-coated tablets andfilm-coated tablets), pills, granules, dispersants, capsules (includingsoft capsules), syrups, emulsions and suspensions. These compositionsare prepared according to conventional methods and contain carriers,diluents or excipients conventionally used in the field of medicinemanufacture. For example, lactose, starch, sucrose, magnesium stearateand the like are used as carriers or excipients for tablets.

Compositions for parenteral administration include, for example,injections and suppositories. Injections include intravenous injections,subcutaneous injections, intradermal injections, muscle injections,instilment injections, etc. Such injections may be prepared bydissolving, suspending or emulsifying the above antibody or salt thereofin an aseptic, aqueous or oily liquid. Examples of aqueous liquids forinjection include physiological saline and isotonic solutions containingglucose and other auxiliary agents. They may be used in combination witha suitable auxiliary solubilizer such as alcohol (e.g. ethanol),polyalcohol (e.g. propylene glycol, polyethylene glycol), nonionicsurfactant [e.g. Polysorbate 80™, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)], etc.). Examples of oily liquids forinjection include sesame oil and soybean oil. They may be used incombination with an auxiliary solubilizer such as benzyl benzoate,benzyl alcohol, etc. Usually, the prepared injections are filled inappropriate ampoules.

It is convenient to formulate the above-described pharmaceuticalcompositions for oral or parenteral administration into unit dosageforms that would give an appropriate dose of the active ingredientExamples of such unit dosage forms include tablets, pills, capsules,injections (ampoules), and suppositories. Usually, each unit of thesedosage forms contains preferably about 5-500 mg of the above-describedantibody. In particular, each unit contains preferably about 5-100 mg ininjections, and each unit in other dosage forms contains preferablyabout 10-250 mg.

The above-described pharmaceutical compositions may contain other activeingredients as long as they do not produce undesirable interaction withthe above-described antibody.

In the specification and drawings of the present application, theabbreviations used for bases, amino acids and so forth are thoserecommended by the IUPAC-IUB Commission on Biochemical Nomenclature orthose conventionally used in the art. Examples of such abbreviations aregiven below. Amino acids, which may have optical isomers are intended torepresent their L-isomer unless otherwise specified.

-   DNA: Deoxyribonucleic acid-   cDNA: Complementary deoxyribonucleic acid-   A: Adenine-   T: Thymine-   G: Guanine-   C: Cytosine-   RNA: Ribonucleic acid-   mRNA: Messenger ribonucleic acid-   dATP: Deoxyadenosine triphosphate-   dTTP: Deoxythymidine triphosphate-   dGTP: Deoxyguanosine triphosphate-   dCTP: Deoxycytidine triphosphate-   ATP: Adenosine triphosphate-   EDTA: Ethylenediaminetetracetic acid-   SDS: Sodium dodecyl sulfate-   Gly: Glycine-   Ala: Alanine-   Val: Valine-   Leu: Leucine-   Ile: Isoleucine-   Ser: Serine-   Thr: Threonine-   Cys: Cysteine-   Met: Methionine-   Glu: Glutamic acid-   Asp: Aspartic acid-   Lys: Lysine-   Arg: Arginine-   His: Histidine-   Phe: Phenylalanine-   Tyr: Tyrosine-   Trp: Tryptophan-   Pro: Proline-   Asn: Asparagine-   Gln: Glutamine-   pGlu: Pyroglutamic acid

The substituents, protective groups and reagents that are frequentlyused in the specification are represented by the followingabbreviations.

-   Me: Methyl group-   Et: Ethyl group-   Bu: Butyl group-   Ph: Phenyl group-   TC: Thiazolidine-4(R)-carboxamide group-   Tos: p-Toluene sulfonyl-   CHO: Formyl-   Bzl: Benzyl-   Cl₂Bzl: 2,6-Dichlorobenzyl-   Bom: Benzyloxymethyl-   Z: Benzyloxycarbonyl-   Cl-Z: 2-Chlorobenzyloxycarbonyl-   Br-Z: 2-Bromobenzyloxycarbonyl-   Boc: t-Butoxycarbonyl-   DNP: Dinitrophenol-   Trt: Trityl-   Bum: t-Butoxymethyl-   Fmoc: N-9-Fluorenylmethyloxycarbonyl-   HOBt: 1-Hydroxybenzotriazole-   HOOBt: 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine-   HONB: 1-Hydroxy-5-norbornene-2,3-dicarboximide-   DCC: N,N′-Dicyclohexylcarbodiimide

The SEQ ID NOS of the SEQUENCE LISTING of the present specificationrepresent the sequences as indicated below.

[SEQ ID NO: 1]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 2]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 3]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 4]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 5]

This shows the base sequence of a cDNA encoding a cubilin partialpeptide obtained in Example 1.

[SEQ ID NO: 6]

This shows the base sequence of a cDNA encoding a cubilin partialpeptide obtained in Example 1.

[SEQ ID NO: 7]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 8]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 9]

This shows the base sequence of a cDNA encoding partial fragment III ofhuman cubilin.

[SEQ ID NO: 10]

This shows the amino acid sequence of partial fragment III of humancubilin.

[SEQ ID NO: 11]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 12]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 13]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 14]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 15]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 16]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 17]

This shows the base sequence of a sense-strand primer used in Example 1.

[SEQ ID NO: 18]

This shows the base sequence of an antisense-strand primer used inExample 1.

[SEQ ID NO: 19]

This shows the amino acid sequence of fragment peptide CUB9-CUB14 thatis a partial fragment of human cubilin.

[SEQ ID NO: 20]

This shows the base sequence of a cDNA encoding fragment peptideCUB7-CUB12 that is a partial fragment of human cubilin.

[SEQ ID NO: 21]

This shows the amino acid sequence of a polypeptide used in Example 5.

[SEQ ID NO: 22]

This shows the base sequence of a cDNA encoding fragment peptideCUB9-CUB14 that is a partial fragment of human cubilin.

A transformant Escherichia coli DH5α/pTB2116 obtained in Example 1 hasbeen deposited at the International Patent Organism Depository, NationalInstitute of Advanced Industrial Science and Technology, located atCentral 6, 1-1 Higashi 1-chome, Tsukuba, Ibaraki under the Accession No.FERM BP-7190 since Jun. 19, 2000, and at the Institute for Fermentation,Osaka (IFO), located at 17-85 Jusanboncho 2-chome, Yodogawa-ku, Osaka,Osaka under the Accession No. IFO 16438 since Jun. 1, 2000.

Another transformant Escherichia coli DH5α/pTB2231 obtained in Example 4has been deposited at the International Patent Organism Depository,National Institute of Advanced Industrial Science and Technology,located at Central 6, 1-1 Higashi 1-chome, Tsukuba, Ibaraki under theAccession No. FERM BP-7607 since May 24, 2001, and at the Institute forFermentation, Osaka (IFO), located at 17-85 Jusanboncho 2-chome,Yodogawa-ku, Osaka, Osaka under the Accession No. IFO 16626 since May17, 2001.

EXAMPLES

The present invention will be described in more detail with reference tothe following Examples. These Examples are provided only forexplanation, and are not intended to limit the scope of the presentinvention.

Example 1

Cloning of cDNAs Encoding Partial Fragments of Human Cubilin

Using a human small intestine-derived cDNA library, cDNAs encodingpartial fragments of human cubilin were cloned by PCR as describedbelow.

A PCR reaction was performed using Marathon Ready cDNA (Clontech)derived from human small intestine, the oligo DNA shown in SEQ ID NO: 1as a sense strand primer and the oligo DNA shown in SEQ ID NO: 2 as ananti-sense strand primer to thereby obtain a cDNA (SEQ ID NO: 5)encoding a partial peptide of human cubilin. A cDNA (SEQ ID NO: 6)encoding a partial peptide of human cubilin was obtained in the samemanner by PCR using the oligo DNA shown in SEQ ID NO: 3 as a sensestrand primer and the oligo DNA shown in SEQ ID NO: 4 as an anti-sensestrand primer. Then, the cDNA having the base sequence as shown in SEQID NO: 5 and the cDNA having the base sequence as shown in SEQ ID NO: 6were mixed. Using this mixture as a template, a PCR reaction wasperformed with the oligo DNA shown in SEQ ID NO: 7 as a sense strandprimer and the oligo DNA shown in SEQ ID NO: 8 as an anti-sense strandprimer. After digestion with restriction enzymes BamHI and NotI, the PCRproduct was inserted into a vector pSecTag2 (Invitrogen) pre-treatedwith the same restriction enzymes. As a result, a 2781 bp cDNA shown inSEQ ID NO: 9 encoding partial fragment III of human cubilin [SEQ ID NO:10; a fragment having the amino acid sequence consisting of the aminoacids from position 1165 to position 2091 of the amino acid sequence ofhuman cubilin mature protein (FIGS. 1 and 2). This fragment correspondsto fragment peptide CUB7-CUB14.] was cloned into the vector that is aneukaryotic expression vector where Igκ signal sequence has been added toan upstream region and Myc and His tags have been added to a downstreamregion.

Similarly, using a cDNA obtained from Caco-2 cell-derived purified mRNAby RT-PCR with random primers, a PCR reaction was performed with theoligo DNA shown in SEQ ID NO: 11 as a sense strand primer and the oligoDNA shown in SEQ ID NO: 12 as an anti-sense strand primer to therebyobtain a cDNA encoding partial fragment I [a fragment having the aminoacid sequence consisting of the amino acids from position 25 to position816 of the amino acid sequence of human cubilin mature protein (FIGS. 1and 2)]. A cDNA encoding partial fragment II [a fragment having theamino acid sequence consisting of the amino acids from position 474 toposition 1390 of the amino acid sequence of human cubilin mature protein(FIGS. 1 and 2)] was obtained in the same manner using the oligo DNAshown in SEQ ID NO: 13 as a sense strand primer and the oligo DNA shownin SEQ ID NO: 14 as an anti-sense strand primer. These cDNAs wereindividually cloned into the above-described expression vector.

Further, using a human small intestine-derived Marathon Ready cDNA(Clontech), a PCR reaction was performed similarly with the oligo DNAshown in SEQ ID NO: 15 as a sense strand primer and the oligo DNA shownin SEQ ID NO: 16 as an anti-sense strand primer to thereby obtain a cDNAencoding partial fragment IV [a fragment having the amino acid sequenceconsisting of the amino acids from position 1852 to position 2804 of theamino acid sequence of human cubilin mature protein (FIGS. 1 and 2)]. AcDNA encoding partial fragment V [a fragment having the amino acidsequence consisting of the amino acids from position 2569 to position3623 of the amino acid sequence of human cubilin mature protein (FIGS. 1and 2)] was obtained in the same manner using the oligo DNA shown in SEQID NO: 17 as a sense strand primer and the oligo DNA shown in SEQ ID NO:18 as an anti-sense strand primer. These cDNAs were individually clonedinto the above-described expression vector.

Further, a plasmid pTB2116 obtained by integrating the cDNA (SEQ ID NO:9) encoding a partial fragment III of human cubilin into pSecTag2 vectoras described above was introduced into Escherichia coli DH5α accordingto publicly known methods to thereby obtain a transformant designatedEscherichia coli DH5α/pTB2116.

Example 2

Expression of Human Cubilin Partial Fragments in COS7 Cells

The expression vector pTB2116 obtained in Example 1 comprising the cDNAhaving the base sequence as shown in SEQ ID NO: 9 was transientlytransfected into COS7 cells using a transfection reagent TransFast(Promega). Subsequently, the cells were transferred to a serum-freemedium to allow them to produce a polypeptide partial fragment III)having the amino acid sequence as shown in SEQ ID NO: 10 in theserum-free medium. With respect to partial fragments I, II, IV and V,corresponding expression vectors were separately transfected into COS7cells in the same manner to allow the resultant cells to producerespective fragments in the serum-free medium. In order to confirmexpression, an aliquot of each culture supernatant was subjected toSDS-PAGE (12.5% gel) under non-reducing conditions. After the gel wastransferred onto a PVDF membrane by a semi-dry method, the fragment ofinterest was detected by Western blotting using anti-Myc monoclonalantibody 9E10 (Sigma). As shown in FIG. 3, it was confirmed thatoverlapping partial fragments I to V of human cubilin were expressed inCOS7 cells and secreted into the culture supernatant.

Example 3

Interaction between Human Apohpoprotein A-I-conjugated Resin and PartialFragments of Human Cubilin

Human apolipoprotein A-I-cross linking Sepharose resin was prepared bymixing human apolipoprotein A-I (Sigma) and swollen CNBr-activatedSepharose (Amersham Pharmacia) at a ratio of 2 mg/l ml in a bindingbuffer (pH 8.3) containing 0.5 M NaCl and 0.1 M NaHCO₃ overnight at 4°C. The resultant human apolipoprotein A-I-linking resin (50 μl) wasmixed with 200 μl each of the culture supernatants obtained in Example 2containing partial fragments I to V, respectively, overnight at 4° C.After removal of the unbound fraction by centrifugation, the reactionmixture was washed with 400 μl of 0.2% bovine albumin-containing D-MEMmedium (Gibco) four times, with D-MEM medium once, and mixed with 20 mMEDTA-containing D-MEM medium. After incubation for 5 min, a fractionthat elutes in an EDTA selective manner was obtained by centrifugation.The unbound fraction and the fraction that elutes in an EDTA selectivemanner were subjected to SDS-PAGE (12.5% gel) separately. The resultantgel was transferred onto a PVDF membrane by a semi-dry method andsubjected to detection using anti-Myc monoclonal antibody 9E10 (Sigma).As shown in FIG. 4, it was revealed that only partial fragment III boundto the human apolipoprotein A-I-linking resin and was eluted with EDTAselectively.

From the above, it was made clear that the human apolipoproteinA-I-binding site of human cubilin is in partial fragment III(corresponding to the fragment peptide CUB7-CUB14).

Example 4

The Apo A-I-Binding Action of CUB9-CUB14 Fragment Peptide

In order to further specify the location of the human apolipoproteinA-I-binding site, a cDNA encoding the amino acid sequence as shown inSEQ ID NO: 19 corresponding to fragment peptide CUB9-CUB14 [having theamino acid sequence consisting of the amino acids from position 1391 toposition 2091 of the amino acid sequence of human cubilin mature protein(FIGS. 1 and 2)] and a cDNA encoding the amino acid sequence as shown inSEQ ID NO: 20 corresponding to fragment peptide CUB7-CUB12 weresub-cloned separately into the expression vector used in Example 1.

The above-mentioned cDNA (SEQ ID NO: 22) encoding human CUB9-CUB14fragment peptide was inserted into pSecTag2 vector. The resultantplasmid pTB2231 was introduced into Escherichia coli DH5α by a publiclyknown method to obtain a transformant designated Escherichia coliDH5α/pTB2231.

These cubilin partial fragments were expressed in culture supernatant ofCOS7 cells in the same manner as in Example 2, and their interactionwith human apolipoprotein A-I-linking resin was examined in the samemanner as in Example 3.

The partial protein containing CUB9-CUB14 fragment peptide bound tohuman apolipoprotein A-I-linking resin (FIG. 5). This result revealedthat the human apolipoprotein A-I-binding site of human cubilin is inCUB9-CUB14 fragment peptide.

Example 5

The Apo A-I-Binding Action of CUB9-CUB14 Fragment Peptide

The cDNA (SEQ ID NO: 22) encoding CUB9-CUB14 fragment peptide wasinserted into a baculovirus for expressing the peptide in insect cells.

Specifically, a cDNA encoding the amino acid sequence as shown in SEQ IDNO: 21 (Myc tag and His tag are added to the C-terminal of CUB9-CUB14fragment peptide) was sub-cloned into pFasBac vector. Using this vector,a baculovirus was constructed according to conventional methods tothereby prepare a system for expressing the fragment peptide in culturesupernatant of HiFive cell, an insect cell.

It was found in the same manner as in Example 4 that the partial proteinhaving CUB9-CUB14 fragment peptide expressed in the culture supernatantbound to human apolipoprotein A-I-linking resin (FIG. 5). This resultrevealed that CUB9-CUB14 fragment peptide expressed in an insect cellalso binds to human apolipoprotein A-I.

Example 6

Purification of CUB9-CUB14 Fragment Peptide

The CUB9-CUB14 fragment peptide expressed in the insect cell obtained inExample 5 was purified using the His tag added to the C-terminal.

Specifically, the culture supernatant was precipitated with 50%saturated ammonium sulfate, and the precipitate was recovered bycentrifugation (12,000 g, 30 min). The resultant precipitate wasdissolved in a buffer containing 20 mM Tris-HCl (pH 7.4), 500 mM NaCl,10 mM imidazole and 0.1% sodium azide. The resultant solution wasapplied to HiTrap Chelating column (Amersham Pharmacia), charged with0.1 M NiSO₄ and equilibrated with the same buffer. After washing withthe same buffer, a linear gradient from 10 mM imidazole to 200 mMimidazole was formed with the buffer to thereby elute CUB9-CUB14fragment peptide.

The thus eluted fractions were subjected to SDS-PAGE (12.5% gel). Theresultant gels stained with CBB are shown in FIG. 6A. The gelstransferred onto PVDF membrane and subjected to detection with anti-Mycmonoclonal antibody 9E10 (Sigma) are shown in FIG. 6B.

From these results, it was found that the eluted fractions containCUB9-CUB14 fragment peptide as a major component.

Example 7

Quantitative Determination of the Binding Between CUB9-CUB14 FragmentPeptide and ApoA-I

The CUB9-CUB14 fragment peptide expressed in the insect cell obtained inExample 5 was dissolved in a buffer containing 20 mM Tris-HCl, 500 mMNaCl, 2 mM CaCl₂ and 0.1% sodium azide to give a final concentration of1-5 μg/ml, and incubated in a fluoroimmunoassay plate (Clini plate Blackenhanced binding; Flow Lab.) containing 100 μl/well of this solution tothereby solidify the peptide. After blocking the non-specific bindingsites with 200 μl/well of SuperBlock TBS (Pierce), the peptide wasincubated with biotin-added human apolipoprotein A-I and, after washing,reacted with streptavidin β-galactosidase. After washing,β-galactosidase activity was measured by conventional methods to therebyquantitatively determine the binding between solidified CUB9-CUB14fragment peptide and human apolipoprotein A-I. For the above bindingreaction and washing, a buffer containing 20 mM Tris-HCl (pH 7.4), 150mM NaCl, 1 mM CaCl₂, 0.1% sodium azide and 5% (v/v) Block-Ace (DainipponPharmaceutical Co., Ltd.) was used.

As shown in FIG. 7, binding reaction dependent on the amount ofsolidified CUB9-CUB14 fragment peptide and dependent on the amount ofbiotin-added human apolipoprotein A-I was recognized. The binding was sostrong that the amount of binding did not change even after washing for2-3 hours. From the binding curve, the approximate affinity (Kd) ofapolipoprotein A-I is judged to be at levels of 20-100 nM.

Example 8

Screening Method

The CUB9-CUB14 fragment peptide expressed in the insect cell obtained inExample 5 was incubated at a concentration of 3 μg/ml in afluoroimmunoassay plate (Clini plate Black enhanced binding; Flow Lab.)containing 100 μl/well of the peptide solution to thereby solidify thepeptide. After blocking the non-specific binding sites with 200 μl/wellof SuperBlock TBS (Pierce), the test compound was incubated withbiotin-added human apolipoprotein A-I (final concentration: 0.1 μg/ml)overnight. After washing, the test compound (100 μl/well) was reactedwith 0.05 U/ml of streptavidin β-galactosidase for 1 hour. Afterwashing, 0.5 mM 4-methylumbelliferyl β-D-galactopyranoside was added tothe reaction solution dissolved in a buffer containing 10 mM K₂HPO₄, 150nM NaCl, 2 mM MgCl₂, 0.1% sodium azide and 0.2% BSA (pH 7.0) andincubated, followed by measurement of excitation at 365 nm andabsorbance at 460 nm. For the above binding reaction and washing, abuffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM CaCl₂, 0.1%sodium azide and 5% (v/v) Block-Ace (Dainippon Pharmaceutical Co., Ltd.)was used.

INDUSTRIAL APPLICABILITY

The polypeptide of the invention (or amide or ester thereof, or saltsthereof) and the DNA of the invention may be used as medicines such astherapeutic and/or prophylactic agent for diabetes, obesity,arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I, or nervousl disorders.

Further, compounds or salts thereof, which are obtainable by a screeningmethod or kit using the polypeptide of the invention, that inhibit thebinding of cubilin to apolipoprotein A-I may be used as medicines suchas therapeutics and/or prophylactics for diseases such as hyperlipemia,hypertriglyceridemia, hypo-high density lipoproteinemia,hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity,arteriosclerosis, myocardial infarction or angina; and compounds orsalts thereof, which are obtainable by a screening method or kit usingthe polypeptide of the invention, that promote the binding of cubilin toapolipoprotein A-I may be used as medicines such as therapeutics and/orprophylactics for diseases such as renal disorders, nephritis,nephropathy, proteinuria, neurological disorders or vitamin B₁₂deficiency.

Further, since the antibody of the invention can specifically recognizethe polypeptide of the invention and cubilin, it may be used inquantitative determination of the polypeptide of the invention in asample solution. Also, the antibody of the invention may be used as amedicine such as a therapeutic and/or prophylactic agent for diseasescaused by excessive expression of the polypeptide of the invention.

The DNA of the invention may be useful as a gene diagnostic agent whenused as a probe, for example.

1. A polypeptide having the ability to bind to apolipoprotein A-I, whichis a partial fragment of cubilin, an amide or ester of said polypeptide,or a salt of said polypeptide.
 2. The polypeptide, amide or esterthereof, or salt thereof of claim 1, wherein the polypeptide ischaracterized by having an amino acid sequence identical orsubstantially identical with the amino acid sequence as shown in SEQ IDNO:
 10. 3. The polypeptide, amide or ester thereof, or salt thereof ofclaim 1, wherein the polypeptide has the amino acid sequence as shown inSEQ ID NO:
 10. 4-5. (canceled)
 6. A DNA comprising a DNA encoding thepolypeptide of claim
 1. 7. The DNA of claim 6, wherein the DNA comprisesthe base sequence as shown in SEQ ID NO:
 9. 8. The DNA of claim 6,wherein the DNA comprises the base sequence as shown in SEQ ID NO: 22.9. A recombinant vector comprising the DNA of claim
 6. 10. Atransformant transformed with the recombinant vector of claim
 9. 11. Amethod of producing the polypeptide, amide or ester thereof, or saltthereof of claim 1, comprising: culturing a transformant transformedwith a recombinant vector comprising a DNA encoding the polypeptide ofclaim 1 and allowing the transformant to produce the polypeptide.
 12. Anantibody to the polypeptide, amide or ester thereof, or salt thereof ofclaim
 1. 13. A method of screening for compounds or salts thereof thatpromote or inhibit the activity of the polypeptide or salt thereof ofclaim 1, wherein the method is characterized by using the polypeptide,amide or ester thereof, or salt thereof of claim 1 in a screening assay.14. A screening kit for compounds or salts thereof that promote orinhibit the activity of the polypeptide, amide or ester thereof, or saltthereof of claim 1, wherein the kit comprises the polypeptide or saltthereof of claim
 1. 15. A compound or salt thereof that promotes orinhibits the activity of a polypeptide having the ability to bind toapolipoprotein A-I, which is a partial fragment of cubilin, amide orester thereof, or salt thereof wherein said compound or salt thereof isobtainable by using the screening method of claim
 13. 16. A medicinecomprising a compound or salt thereof that promotes or inhibits theactivity of a polypeptide having the ability to bind to apolipoproteinA-I, which is a partial fragment of cubilin, amide or ester thereof, orsalt thereof wherein said compound or salt thereof is obtainable byusing the screening method of claim
 13. 17. A prophylactic and/ortherapeutic agent for renal disorders, nephritis, nephropathy,proteinuria, nervous disorders or vitamin B₁₂ deficiency, comprising acompound or salt thereof that promotes or inhibits the activity of apolypeptide having the ability to bind to apolipoprotein A-I, which is apartial fragment of cubilin, amide or ester thereof, or salt thereofwherein said compound or salt thereof is obtainable by using thescreening method of claim
 13. 18. A prophylactic and/or therapeuticagent for hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia,diabetes, obesity, arteriosclerosis, myocardial infarction or angina,comprising a compound or salt thereof that promotes or inhibits theactivity of a polypeptide having the ability to bind to apolipoproteinA-I, which is a partial fragment of cubilin, amide or ester thereof, orsalt thereof wherein said compound or salt thereof is obtainable byusing the screening method of claim
 13. 19. A pharmaceutical compositioncomprising the polypeptide, amide or ester thereof, or salt thereof ofclaim 1 and a pharmacologically acceptable carrier, excipient ordiluent.
 20. A prophylactic and/or therapeutic agent for diabetes,obesity, arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-highdensity lipoproteinemia, hypoapolipoproteinemia A-I or nervousdisorders, comprising the polypeptide, amide or ester thereof, or saltthereof of claim
 1. 21. A diagnostic agent that is characterized by theuse of the DNA of claim
 6. 22. A diagnostic agent comprising theantibody of claim
 12. 23. A method for preventing and/or treating renaldisorders, nephritis, nephropathy, proteinuria, nervous disorders orvitamin B₁₂ deficiency in a mammal, comprising a compound or saltthereof that promotes or inhibits the activity of a polypeptide havingthe ability to bind to apolipoprotein A-I, which is a partial fragmentof cubilin, amide or ester thereof, or salt thereof wherein saidcompound or salt thereof is obtainable by using the screening method ofclaim
 13. 24. A method for preventing and/or treating hyperlipemia,hypertriglyceridemia, hypo-high density lipoproteinemia,hypoapolipoproteinemia A-I, after meal hyperlipemia, diabetes, obesity,arteriosclerosis, myocardial infarction or angina, characterized byadministering to a mammal an effective amount of a compound or saltthereof that promotes or inhibits the activity of a polypeptide havingthe ability to bind to apolipoprotein A-I, which is a partial fragmentof cubilin, amide or ester thereof, or salt thereof wherein saidcompound or salt thereof is obtainable by using the screening method ofclaim
 13. 25. A method for manufacturing a prophylactic and/ortherapeutic agent for treating or preventing, in a mammal in needthereof, hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I, after meal hyperlipemia,diabetes, obesity, arteriosclerosis, myocardial infarction or angina,comprising combining a therapeutic amount of a compound or salt thereofthat promotes or inhibits the activity of a polypeptide having theability to bind to apolipoprotein A-I, which is a partial fragment ofcubilin, amide or ester thereof, or salt thereof, wherein said compoundor salt thereof is obtainable by using the screening method of claim 13with a pharmacologically acceptable carrier, excipient or diluent.
 26. Amethod for preventing and/or treating diabetes, obesity,arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders,characterized by administering to a mammal an effective amount of thepolypeptide, amide or ester thereof, or salt thereof of claim
 1. 27. Amethod for manufacturing a prophylactic and/or therapeutic agent fortreating or preventing, in a mammal in need thereof, diabetes, obesity,arteriosclerosis, hyperlipemia, hypertriglyceridemia, hypo-high densitylipoproteinemia, hypoapolipoproteinemia A-I or nervous disorders,comprising combining a therapeutic amount of a compound or salt thereofthat promotes or inhibits the activity of a polypeptide having theability to bind to apolipoprotein A-I, which is a partial fragment ofcubilin, amide or ester thereof, or salt thereof with apharmacologically acceptable carrier, excipient or diluent.